Process of extracting oil from thin stillage

ABSTRACT

The present invention relates to processes of recovering oil after liquefaction and/or from thin stillage and/or syrup/evaporated centrate from a fermentation product production process by adding a thermostable protease to the whole stillage, thin stillage and/or syrup.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.17/934,465 filed Sep. 22, 2022, which is a continuation of U.S.application Ser. No. 17/147,623 filed Jan. 13, 2021, now U.S. Pat. No.11,505,765, which is a continuation of U.S. application Ser. No.16/449,703 filed Jun. 24, 2019, now U.S. Pat. No. 10,920,172, which is acontinuation of U.S. application Ser. No. 15/956,274 filed Apr. 18,2018, now U.S. Pat. No. 10,731,104, which is a continuation of U.S.application Ser. No. 14/901,504 filed Dec. 28, 2015, now U.S. Pat. No.10,035,973, which is a 35 U.S.C. 371 national application ofPCT/US2014/043392 filed Jun. 20, 2014, which claims priority or thebenefit under 35 U.S.C. 119 of U.S. provisional application Nos.61/838,650, 61/863,727, 61/943,794 and 61/991,866 filed Jun. 24, 2013,Aug. 8, 2013, Feb. 24, 2014 and May 12, 2014, respectively, the contentsof which are fully incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to processes of extracting/recovering oilfrom liquefied material as well as thin stillage and/or syrup at thebackend of a fermentation product production process based onstarch-containing material.

BACKGROUND OF THE INVENTION

Fermentation products, such as ethanol, are typically produced by firstgrinding starch-containing material in a dry-grind or wet-millingprocess, then degrading the material into fermentable sugars usingenzymes and finally converting the sugars directly or indirectly intothe desired fermentation product using a fermenting organism. Liquidfermentation products are recovered from the fermented mash (oftenreferred to as “beer mash”), e.g., by distillation, which separate thedesired fermentation product from other liquids and/or solids. Theremaining fraction is referred to as “whole stillage”. The wholestillage is dewatered and separated into a solid and a liquid phase,e.g., by centrifugation. The solid phase is referred to as “wet cake”(or “wet grains”) and the liquid phase (supernatant) is referred to as“thin stillage”. Wet cake and thin stillage contain about 35 and 7%solids, respectively. Dewatered wet cake is dried to provide “DistillersDried Grains” (DDG) used as nutrient in animal feed. Thin stillage istypically evaporated to provide condensate and syrup or mayalternatively be recycled directly to the slurry tank as “backset”.Condensate may either be forwarded to a methanator before beingdischarged or may be recycled to the slurry tank. The syrup may beblended into DDG or added to the wet cake before drying to produce DDGS(Distillers Dried Grain with Solubles). An increasing number of ethanolplants extract oil from the thin stillage and/or syrup/evaporatedcentrate as a by-product for use in biodiesel production or otherbiorenewable products.

Much of the work in oil recovery/extraction from fermentation productproduction processes has focused on improving the extractability of theoil from the thin stillage. Effective removal of oil is oftenaccomplished by hexane extraction. However, the utilization of hexaneextraction has not seen widespread application due to the high capitalinvestment required. Therefore, other processes that improve oilextraction from fermentation product production processes have beenexplored.

WO 2011/126897 (Novozymes) discloses processes of recovering oil byconverting starch-containing materials into dextrins with alpha-amylase;saccharifying with a carbohydrate source generating enzyme to formsugars; fermenting the sugars using fermenting organism; wherein thefermentation medium comprises a hemicellulase; distilling thefermentation product to form whole stillage; separating the wholestillage into thin stillage and wet cake; and recovering oil from thethin stillage. The fermentation medium may further comprise a protease.

It is an object of the present invention to provide improved processesfor increasing the amount of recoverable oil from fermentation productproduction processes.

SUMMARY OF THE INVENTION

The object of the present invention is to provide improved processes ofextracting or recovering oil at the backend of a fermentation productproduction process, such as especially an ethanol production process.

Therefore, in the first aspect the invention relates to processes ofrecovering oil, comprising

-   -   (a) converting a starch-containing material into dextrins with        an alpha-amylase; optionally recovering oil during and/or after        step (a)    -   (b) saccharifying the dextrins using a carbohydrate source        generating enzyme to form a sugar;    -   (c) fermenting the sugar in a fermentation medium into a        fermentation product using a fermenting organism;    -   (d) recovering the fermentation product to form a whole        stillage;    -   (e) separating the whole stillage into thin stillage and wet        cake;    -   (e′) optionally concentrating the thin stillage into syrup;    -   (f) recovering oil from the thin stillage and/or optionally the        syrup, wherein a protease having a thermostability value of more        than 20% determined as Relative Activity at 80° C./70° C. is        present and/or added in step (a) or any one of steps (d)-(e′).

In an embodiment the protease may have a thermostability of above 90%,above 100% at 85° C. as determined using the Zein-BCA assay as disclosedin Example 2.

In a preferred embodiment the protease is present in and/or added instarch-containing material converting step (a). In an embodiment thetemperature in step (a) is above the initial gelatinization temperature,such as between 80-90° C., such as around 85° C. Steps (b) and (c) maybe carried out simultaneously or sequentially. In an embodiment steps(a), (b) and (c) are carried our simultaneously or sequentially. Whensteps (a), (b) and (c) are carried out simultaneously, the temperatureis below the initial gelatinization temperature, such as between 25-40°C., preferably around 32° C. in case of producing fermentation productssuch as ethanol.

In a preferred embodiment the protease is present in or added to thewhole stillage in step (d) and/or to the thin stillage after separatingwhole stillage into thin stillage and wet cake in step (e), and/or tothe syrup in step (e′). In such embodiments step (a) may be carried outat temperatures at or above the initial gelatinization temperature, suchas between 80-90° C., or below the initial gelatinization temperature,such as between 25-40° C. In a preferred embodiment oil is recoveredfrom the thin stillage and/or syrup/evaporated centrate, e.g., byextraction, such as hexane extraction or by using another oil recoverytechnology well-known in the art.

The protease may be any protease having a thermostability value, asdefined herein, of more than 20% determined as Relative Activity.Determination of “Relative Activity” and “Remaining Activity” isdetermined as described in Example 1. In an embodiment the protease hasa thermostability value of more than 30%, more than 40%, more than 50%,more than 60%, more than 70%, more than 80%, more than 90% more than100%, such as more that 105%, such as more than 110%, such as more than115%, such as more than 120% determined as Relative Activity at 80°C./70° C.

In an embodiment the protease may have a thermostability of above 90%,above 100% at 85° C. as determined using the Zein-BCA assay as disclosedin Example 2.

In an embodiment the protease is a thermostable variant of the parentmetallo protease derived Thermoascus aurantiacus shown in SEQ ID NO: 3herein, classified as EC 3.4.24.39, or one having a sequence identitythereto of at least 60%, at least 70%, at least 80%, at least 90%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%.Examples of protease variants are disclosed in the section “Proteases”below and in the Examples below. In a preferred embodiment the proteasevariant is selected from the group comprising the followingsubstitutions:

-   -   A27K+D79L+Y82F+S87G+D104P+A112P+A126V+D142L;    -   D79L+Y82F+S87G+A112P+D142L;    -   Y82F+S87G+S70V+D79L+D104P+A112P+D142L;    -   Y82F+S87G+D79L+D104P+A112P+A126V+D142L (using SEQ ID NO: 3 for        numbering).

All of these protease variants have a higher thermostability value (asdefined herein) than the wild-type parent protease shown in SEQ ID NO: 3herein.

In an additional embodiment the protease is a filamentous fungus, e.g.,a protease classified as EC 3.4.23.23, such as derived from a strain ofRhizomucor, such as Rhizomucor miehei, such as the protease shown in SEQID NO: 9 or one having a sequence identity thereto of at least 60%, atleast 70%, at least 80%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%.

In another preferred embodiment the protease is a thermostable proteasederived from the bacterium, e.g., classified as EC 3.4.21.62, such asPyrococcus furiosus, such as the protease shown in SEQ ID NO: 4 or onehaving a sequence identity thereto of at least 60%, at least 70%, atleast 80%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%.

In an additional embodiment the protease is a bacterial serine protease,such as derived from a strain of Thermobifida, such as Thermobifidafusca, such as the protease shown in SEQ ID NO: 10 or one having asequence identity thereto of at least 60%, at least 70%, at least 80%,at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%.

According to the invention the oil may be extracted/recovered from theliquefied starch-containing material during and/or after step (a),before saccharification in step (b). Therefore, the invention alsorelates to processes of recovering oil, comprising

-   -   (a) converting a starch-containing material into dextrins with        an alpha-amylase;        -   recovering oil during and/or after step (a)    -   (b) saccharifying the dextrins using a carbohydrate source        generating enzyme to form a sugar;    -   (c) fermenting the sugar in a fermentation medium into a        fermentation product using a fermenting organism; wherein a        protease having a thermostability value of more than 20%        determined as Relative Activity at 80° C./70° C. is present        and/or added in step (a).

In such embodiment oil may not be extracted/recovered at the back-end asdefined herein. However, in an embodiment oil is extracted both duringand/or after step (a) and from the thin stillage and/or optionally thesyrup/evaporated centrate.

In another aspect the invention relates to the use of a protease havinga thermostability value of more than 20%, more than 30%, more than 40%,more than 50%, more than 60%, more than 70%, more than 80%, more than90% more than 100%, such as more that 105%, such as more than 110%, suchas more than 115%, such as more than 120% determined as RelativeActivity at 80° C./70° C. for oil recovery from thin stillage and/orsyrup at the backend of a fermentation product production process basedon starch-containing material.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 schematically shows an ethanol production process. Oil may berecovered/extracted during and/or after liquefaction (step (a)), fromthe thin stillage and/or the syrup/centrate. The boxes in the figureindicate where oil may be recovered/extracted.

FIG. 2 shows the % oil extracted from the whole stillage for Control (Noprotease), Protease PF, Protease RH, Protease TA and Protease TA 196,Protease TF.

FIG. 3 shows the % oil extracted from liquefied corn mash for Controland Protease PF.

FIG. 4 shows the % oil extraction from syrup for Control (No protease),Protease PF, Protease RH, Protease TA and Protease TA 196, Protease TF.

FIG. 5 shows % oil in the liquefied corn mash (liquefact) for Control(No Protease), Protease PF, Protease RH, Protease TA, Protease TA196 andProtease TF.

DETAILED DESCRIPTION OF THE INVENTION

The object of the present invention is to provide improved processes ofextracting or recovering oil at the backend of a fermentation productproduction process, such as especially an ethanol production process.

Therefore, in the first aspect the invention relates to processes ofrecovering oil, comprising

-   -   (a) converting a starch-containing material into dextrins with        an alpha-amylase; optionally recovering oil during and/or after        step (a),    -   (b) saccharifying the dextrins using a carbohydrate source        generating enzyme to form a sugar;    -   (c) fermenting the sugar in a fermentation medium into a        fermentation product using a fermenting organism;    -   (d) recovering the fermentation product to form a whole        stillage;    -   (e) separating the whole stillage into thin stillage and wet        cake;    -   (e′) optionally concentrating the thin stillage into syrup;    -   (f) recovering oil from the thin stillage and/or optionally the        syrup, wherein a protease having a thermostability value of more        than 20% determined as Relative Activity at 80° C./70° C. is        present and/or added in step (a) or during any one of steps        (d)-(e′). In an embodiment the protease may have a        thermostability of above 90%, above 100% at 85° C. as determined        using the Zein-BCA assay as disclosed in Example 2.

In a preferred embodiment the protease is present in or added instarch-containing material converting step (a). In an embodiment thetemperature is above the initial gelatinization temperature, such asbetween 80-90° C., such as around 85° C. In an embodiment step (a) iscarried out as a liquefaction step followed by steps (b) and (c) carriedout either simultaneously or sequentially. In a preferred embodimentsteps (b) and (c) are carried out as a simultaneous saccharification andfermentation step (i.e., SSF).

In a preferred embodiment the oil recovering process of the inventioncomprises:

-   -   (a) converting a starch-containing material into dextrins with        an alpha-amylase at a temperature above the initial        gelatinization temperature;    -   (b) saccharifying the dextrins using a carbohydrate source        generating enzyme to form a sugar;    -   (c) fermenting the sugar in a fermentation medium into a        fermentation product using a fermenting organism;    -   (d) recovering the fermentation product to form a whole        stillage;    -   (e) separating the whole stillage into thin stillage and wet        cake;    -   (e′) optionally concentrating the thin stillage into syrup;    -   (f) recovering oil from the thin stillage and/or optionally the        syrup, wherein a protease having a thermostability value of more        than 20% determined as Relative Activity at 80° C./70° C. is        present and/or added during step (a) or any of claims (d)-(e′).

In another preferred embodiment the oil recovery process of theinvention comprises:

-   -   (a) converting a starch-containing material into dextrins with        an alpha-amylase at a temperature below the initial        gelatinization temperature;    -   (b) saccharifying the dextrins using a carbohydrate source        generating enzyme to form a sugar;    -   (c) fermenting the sugar in a fermentation medium into a        fermentation product using a fermenting organism;    -   (d) recovering the fermentation product to form a whole        stillage;    -   (e) separating the whole stillage into thin stillage and wet        cake;    -   (e′) optionally concentrating the thin stillage into syrup;    -   (f) recovering oil from the thin stillage and/or optionally the        syrup, wherein a protease having a thermostability value of more        than 20% determined as Relative Activity at 80° C./70° C. is        present and/or added during any of step (d)-(e′).

In an embodiment steps (a), (b), and (c) are carried out simultaneously.This is typically done at a temperature below the initial gelatinizationtemperature, i.e. raw starch hydrolysis process (RSH). However, steps(a), (b), and (c) may also be carried out sequentially. In suchembodiments step (a) may be carried out at temperatures at or above theinitial gelatinization temperature, such as between 80-90° C., or belowthe initial gelatinization temperature, such as between 25-40° C., suchas around 32° C.

The term “initial gelatinization temperature” means the lowesttemperature at which gelatinization of the starch commences. Starchheated in water begins to gelatinize between 50° C. and 75° C.; theexact temperature of gelatinization depends on the specific starch, andcan readily be determined by the skilled artisan. Thus, the initialgelatinization temperature may vary according to the plant species, tothe particular variety of the plant species as well as with the growthconditions. In the context of this invention the initial gelatinizationtemperature of a given starch-containing material is the temperature atwhich birefringence is lost in 5% of the starch granules using themethod described by Gorinstein. S. and Lii. C., Starch/Stärke, Vol. 44(12) pp. 461-466 (1992).

In an embodiment the protease is present and/or added at the backend ofa fermentation product production process, such as an ethanol productionprocess, preferably to the whole stillage and/or syrup/evaporatedcentrate, including a conventional ethanol product production process,which included a liquefaction step, e.g., step (a) done at hightemperatures, such as at temperatures at or above the initialgelatinization temperatures, such as at temperatures between 80-90° C.,at a pH between 4.5 and 6.0, followed by simultaneous saccharificationand fermentation (e.g., steps (b) and (c)) done a temperature between25-40° C., such as around 32° C., if the fermentation product is ethanolor the like.

In another embodiment the protease is present and/or added at thebackend of a fermentation product production process, such as an ethanolproduction process, preferably to the whole stillage and/or syrup, wheregranular starch is saccharified and fermented simultaneously attemperatures below the initial gelatinization temperatures, such as attemperatures between 25-40° C., at a pH between 4.5 and 6.0, i.e., steps(a) (b), and (c) are carried out simultaneously.

In a preferred embodiment the protease is present in or added to thewhole stillage in step (d) and/or to the thin stillage after separatingthe whole stillage into thin stillage and wet cake in step (e), and/orto the syrup in step (e′). In a preferred embodiment oil is recoveredfrom the thin stillage and/or syrup/evaporated centrate, e.g., byextraction, such as by hexane extraction or by using another oilrecovery technology well-known in the art.

According to the invention the oil may be extracted from the liquefiedstarch-containing material during and/or after step (a), such as beforesaccharification in step (b). Therefore, the invention also relates toprocesses of recovering oil, comprising

-   -   (b) converting a starch-containing material into dextrins with        an alpha-amylase;        -   recovering oil during and/or after step (a)    -   (b) saccharifying the dextrins using a carbohydrate source        generating enzyme to form a sugar;    -   (c) fermenting the sugar in a fermentation medium into a        fermentation product using a fermenting organism; wherein a        protease having a thermostability value of more than 20%        determined as Relative Activity at 80° C./70° C. is present        and/or added in step (a).

In such embodiment oil may not be extracted at the back-end as definedherein. However, in an embodiment oil is extracted both during and/orafter step (a) and from the thin stillage and/or optionally thesyrup/evaporated centrate.

The protease may be any protease having a thermostability value, asdefined herein, of more than 20% and the Example 1.

In an embodiment the protease has a thermostability value of more than30%, more than 40%, more than 50%, more than 60%, more than 70%, morethan 80%, more than 90% more than 100%, such as more that 105%, such asmore than 110%, such as more than 115%, such as more than 120%determined as Relative Activity at 80° C./70° C.

In one embodiment the protease is a thermostable variant of the metalloprotease derived from Thermoascus aurantiacus shown in SEQ ID NO: 3herein, or one having a sequence identity thereto of at least 90%, andwherein the protease has a thermostability value of more than 30%, morethan 40%, more than 50%, more than 60%, more than 70%, more than 80%,more than 90% more than 100%, such as more that 105%, such as more than110%, such as more than 115%, such as more than 120% determined asRelative Activity at 80° C./70° C.

In one embodiment the protease is a thermostable variant of the metalloprotease derived from Thermoascus aurantiacus shown in SEQ ID NO: 3herein, or one having a sequence identity thereto of at least 95%, andwherein the protease has a thermostability value of more than 30%, morethan 40%, more than 50%, more than 60%, more than 70%, more than 80%,more than 90% more than 100%, such as more that 105%, such as more than110%, such as more than 115%, such as more than 120% determined asRelative Activity at 80° C./70° C.

In one embodiment the protease is a thermostable variant of the metalloprotease derived from Thermoascus aurantiacus shown in SEQ ID NO: 3herein, or one having a sequence identity thereto of at least 99%, andwherein the protease has a thermostability value of more than 30%, morethan 40%, more than 50%, more than 60%, more than 70%, more than 80%,more than 90% more than 100%, such as more that 105%, such as more than110%, such as more than 115%, such as more than 120% determined asRelative Activity at 80° C./70° C.

In an embodiment the protease is a thermostable variant of the metalloprotease derived Thermoascus aurantiacus shown in SEQ ID NO: 3 herein,or one having a sequence identity thereto of at least 60%, at least 70%,at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%. Examples of protease variants are disclosed inthe section “Proteases” below and in the Examples below. In a preferredembodiment the protease variant is selected from the group of variantscomprising the following substitutions:

-   -   A27K+D79L+Y82F+S87G+D104P+A112P+A126V+D142L;    -   D79L+Y82F+S87G+A112P+D142L;    -   Y82F+S87G+S70V+D79L+D104P+A112P+D142L;    -   Y82F+S87G+D79L+D104P+A112P+A126V+D142L (using SEQ ID NO: 3 for        numbering).

All of these protease variants have higher thermostability value (asdefined herein) than the wild-type parent protease shown in SEQ ID NO: 3herein.

In an additional embodiment the protease is a filamentous fungus, e.g.,derived from a strain of Rhizomucor, such as Rhizomucor miehei, such asthe protease shown in SEQ ID NO: 9 or one having a sequence identitythereto of at least 60%, at least 70%, at least 80%, at least 90%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%.

In one embodiment the protease is derived from a strain of Rhizomucormiehei, such as the protease shown in SEQ ID NO: 9 herein, or one havinga sequence identity thereto of at least 90%, and wherein the proteasehas a thermostability value of more than 30%, more than 40%, more than50%, more than 60%, more than 70%, more than 80%, more than 90% morethan 100%, such as more that 105%, such as more than 110%, such as morethan 115%, such as more than 120% determined as Relative Activity at 80°C./70.

In one embodiment the protease is derived from a strain of Rhizomucormiehei, such as the protease shown in SEQ ID NO: 9 herein, or one havinga sequence identity thereto of at least 95%, and wherein the proteasehas a thermostability value of more than 30%, more than 40%, more than50%, more than 60%, more than 70%, more than 80%, more than 90% morethan 100%, such as more that 105%, such as more than 110%, such as morethan 115%, such as more than 120% determined as Relative Activity at 80°C./70.

In one embodiment the protease is derived from a strain of Rhizomucormiehei, such as the protease shown in SEQ ID NO: 9 herein, or one havinga sequence identity thereto of at least 99%, and wherein the proteasehas a thermostability value of more than 30%, more than 40%, more than50%, more than 60%, more than 70%, more than 80%, more than 90% morethan 100%, such as more that 105%, such as more than 110%, such as morethan 115%, such as more than 120% determined as Relative Activity at 80°C./70.

In another preferred embodiment the protease is a thermostable proteasederived from the bacterium Pyrococcus furiosus, such as the proteaseshown in SEQ ID NO: 4, or one having sequence identity thereto of atleast 60%, at least 70%, at least 80%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%.

The Pyrococcus furiosus protease shown in SEQ ID NO: 4 herein is athermostable bacterial protease. A commercial Pyrococcus furiosusprotease product (Pfu S) from Takara Bio Inc. (Japan) have been found tohave a thermostability value of 110% (80° C./70° C.) and 103% (90°C./70° C.) at pH 4.5 determined as described in Example 1 herein.

In one embodiment the protease is a thermostable protease derived fromthe bacterium Pyrococcus furiosus, such as the protease shown in SEQ IDNO: 4 herein, or one having a sequence identity thereto of at least 90%,and wherein the protease has a thermostability value of more than 30%,more than 40%, more than 50%, more than 60%, more than 70%, more than80%, more than 90% more than 100%, such as more that 105%, such as morethan 110%, such as more than 115%, such as more than 120% determined asRelative Activity at 80° C./70.

In one embodiment the protease is a thermostable protease derived fromthe bacterium Pyrococcus furiosus, such as the protease shown in SEQ IDNO: 4 herein, or one having a sequence identity thereto of at least 95%,and wherein the protease has a thermostability value of more than 30%,more than 40%, more than 50%, more than 60%, more than 70%, more than80%, more than 90% more than 100%, such as more that 105%, such as morethan 110%, such as more than 115%, such as more than 120% determined asRelative Activity at 80° C./70.

In one embodiment the protease is a thermostable protease derived fromthe bacterium Pyrococcus furiosus, such as the protease shown in SEQ IDNO: 4 herein, or one having a sequence identity thereto of at least 99%,and wherein the protease has a thermostability value of more than 30%,more than 40%, more than 50%, more than 60%, more than 70%, more than80%, more than 90% more than 100%, such as more that 105%, such as morethan 110%, such as more than 115%, such as more than 120% determined asRelative Activity at 80° C./70.

In an additional embodiment the protease is a bacterial serine protease,such as derived from a strain of Thermobifida, such as Thermobifidafusca, such as the protease shown in SEQ ID NO: 10 or one having asequence identity thereto of at least 60%, at least 70%, at least 80%,at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%.

In one embodiment the protease is derived from a strain of Thermobifida,such as Thermobifida fusca, such as the protease shown in SEQ ID NO: 10herein, or one having a sequence identity thereto of at least 90%, andwherein the protease has a thermostability value of more than 30%, morethan 40%, more than 50%, more than 60%, more than 70%, more than 80%,more than 90% more than 100%, such as more that 105%, such as more than110%, such as more than 115%, such as more than 120% determined asRelative Activity at 80° C./70.

In one embodiment the protease is derived from a strain of Thermobifida,such as Thermobifida fusca, such as the protease shown in SEQ ID NO: 10herein, or one having a sequence identity thereto of at least 95%, andwherein the protease has a thermostability value of more than 30%, morethan 40%, more than 50%, more than 60%, more than 70%, more than 80%,more than 90% more than 100%, such as more that 105%, such as more than110%, such as more than 115%, such as more than 120% determined asRelative Activity at 80° C./70.

In one embodiment the protease is derived from a strain of Thermobifida,such as Thermobifida fusca, such as the protease shown in SEQ ID NO: 10herein, or one having a sequence identity thereto of at least 99%, andwherein the protease has a thermostability value of more than 30%, morethan 40%, more than 50%, more than 60%, more than 70%, more than 80%,more than 90% more than 100%, such as more that 105%, such as more than110%, such as more than 115%, such as more than 120% determined asRelative Activity at 80° C./70.

When step (a) is carried out as a liquefaction step at hightemperatures, i.e., above the initial gelatinization temperature, suchas at temperatures between 80-90° C., such as around 85° C., thealpha-amylase may be a bacterial alpha-amylase. In an embodiment the pHin step (a) is from 4-7, preferably 4.5-6.

In an embodiment a jet-cooking step is carried out before in step (a).Jet-cooking may be carried out at a temperature between 95-140° C. forabout 1-15 minutes, preferably for about 3-10 minutes, especially aroundabout 5 minutes.

In a preferred embodiment a process of the invention further comprises,before step (a), the steps of:

-   -   i) reducing the particle size of the starch-containing material,        preferably by dry milling;    -   ii) forming a slurry comprising the starch-containing material        and water.

In a preferred embodiment the bacterial alpha-amylase is derived fromthe genus Bacillus, such as a strain of Bacillus stearothermophilus, inparticular a variant of a Bacillus stearothermophilus alpha-amylase,such as the one shown in SEQ ID NO: 3 in WO 99/019467 or SEQ ID NO: 1herein, in particular a Bacillus stearothermophilus alpha-amylasetruncated to have from 485-495 amino acids, such as around 491 aminoacids.

In a preferred embodiment the bacterial alpha-amylase is selected fromthe group of Bacillus stearothermophilus alpha-amylase variantscomprising a double deletion, such as I181*+G182*, or I181*+G182*+N193F(using SEQ ID NO: 1 for numbering).

In a preferred embodiment the bacterial alpha-amylase is selected fromthe group of Bacillus stearothermophilus alpha-amylase variants:

-   -   I181*+G182*+N193F+E129V+K177L+R179E;    -   181*+G182*+N193F+V59A+Q89R+E129V+K177L+R179E+H208Y+K220P+N224L+Q254S;    -   I181*+G182*+N193F+V59A Q89R+E129V+K177L+R179E+Q254S+M284V; and    -   I181*+G182*+N193F+E129V+K177L+R179E+K220P+N224L+S242Q+Q254S        (using SEQ ID NO: 1 for numbering).

The parent Bacillus stearothermophilus alpha-amylase may be the oneshown in SEQ ID NO: 1 or may be one having sequence identity thereto ofat least 60%, at least 70%, at least 80%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%.

A Bacillus stearothermophilus alpha-amylase variant may be a variant ofthe one shown in SEQ ID NO: 1 or may be one having sequence identitythereto of at least 60%, at least 70%, at least 80%, at least 90%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, butless than 100%. In an embodiment the Bacillus stearothermophilusalpha-amylase variant has from 1-12 mutations, such as 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12 mutations, compared to the parent alpha-amylase,especially the parent alpha-amylase shown in SEQ ID NO: 1.

Step (a) is followed by saccharification of dextrins in step (b).However, a process of the invention may further comprise apre-saccharification step, i.e., after step (a), but beforesaccharification step (b), carried out for 40-90 minutes at atemperature between 30-65° C.

According to the invention saccharification is carried out at atemperature from 20-75° C., preferably from 40-70° C., such as around60° C., and at a pH between 4 and 5.

In a preferred embodiment fermentation step (c) or simultaneoussaccharification and fermentation (SSF) (i.e., combined steps (b) and(c)) are carried out carried out at a temperature from 25° C. to 40° C.,such as from 28° C. to 35° C., such as from 30° C. to 34° C., preferablyaround about 32° C. In an embodiment fermentation step (c) orsimultaneous saccharification and fermentation (SSF) (i.e., combinedsteps (b) and (c)) are ongoing for 6 to 120 hours, in particular 24 to96 hours. In an embodiment the starch-containing material convertingstep (a), saccharification step (b) and fermentation step (c) arecarried out simultaneously or sequentially. In an embodiment thestarch-containing material converting step (a) is carried out at atemperature below the initial gelatinization temperature, preferablyfrom 20-60° C., such as 25-40° C., such as around 30-34° C., such asaround 32° C. In an embodiment the starch-containing material isconverted to dextrins and the dextrins are saccharified to a sugar bytreating the starch-containing material with an alpha-amylase andglucoamylase below the initial gelatinization temperature of thestarch-containing material. In an embodiment the conversion of thestarch-containing material to dextrins, the saccharification of thedextrins to sugars, and the fermentation of the sugars are carried outin a single step (i.e., raw starch hydrolysis step). When the process ofthe invention is carried out as a raw starch hydrolysis process (i.e.,single step process or no-cook process) the glucoamylase may preferablybe derived from a strain of Trametes, such as a strain of Trametescingulata, or a strain of Athelia, such as a strain of Athelia rolfsii.Preferred alphaamylases used in a raw starch hydrolysis process includealpha-amylases derived from a strain Rhizomucor, such as a strain ofRhizomucor pusillus, such as a Rhizomucor pusillus alpha-amylase with astarch-binding domain (SBD), such as a Rhizomucor pusillus alpha-amylasewith Aspergillus niger glucoamylase linker and SBD. Generally thestarch-containing material in raw starch hydrolysis processes (i.e.,no-cook processes) are granular starch. Said granular starch may bereduced the particle size, preferably by milling, to from 0.05 to 3.0mm, preferably 0.1-0.5 mm. Also the sugar level, such as glucose level,may be kept below 6 wt.-%, preferably below about 3 wt.-%, preferablybelow about 2 wt.-%, more preferred below about 1 wt.-%., even morepreferred below about 0.5%, or even more preferred 0.25% wt.-%, such asbelow about 0.1 wt.-%. The pH may be from 4-7, preferably 4.5-6.0, whenconversion of the starch-containing material to dextrins, thesaccharification of the dextrins to a sugar, and the fermentation of thesugar are carried out in a single step.

If the process of the invention is carried out as a conventional process(i.e., step (a) is carried out as a liquefaction step at a temperatureabove the gelatinization temperature) the carbohydrate-source generatingenzyme used in step (b) is preferably a glucoamylase derived fromAspergillus, preferably A. niger, A. awamori, or A. oryzae; or a strainof Trichoderma, preferably Trichoderma reesei; or a strain ofTalaromyces, preferably Talaromyces emersonii, or a strain ofPycnoporus, or a strain of Gloephyllum.

Examples of other suitable glucoamylase can be found below in the“Glucoamylases” section below.

Generally the starch-containing material in step (a), including granularstarch, contains 20-55 wt.-% dry solids, preferably 25-40 wt.-% drysolids, more preferably 30-35% dry solids.

In an embodiment of the invention the protease is present in or added tothe whole stillage in step (d) and/or the thin stillage, in step (e) orafter separation in step (e), and/or the syrup in step (e′). Separation(i.e. dewatering) in step (e) may be carried out by centrifugation,preferably a decanter centrifuge, filtration, preferably using a filterpress, a screw press, a plate-and-frame press, a gravity thickener ordecker or any other separation technology known in the art. In anembodiment the starch-containing material is cereal. In an embodimentthe starch-containing material is selected from the group consisting ofcorn, wheat, barley, cassava, sorghum, rye, potato, beans, milo, peas,rice, sago, sweet potatoes, tapioca, or any combination thereof.

The (desired) fermentation product may in an embodiment be selected fromthe group consisting of alcohols (e.g., ethanol, methanol, butanol,1,3-propanediol); organic acids (e.g., citric acid, acetic acid,itaconic acid, lactic acid, gluconic acid, gluconate, lactic acid,succinic acid, 2,5-diketo-D-gluconic acid); ketones (e.g., acetone);amino acids (e.g., glutamic acid); gases (e.g., H2 and CO2), and morecomplex compounds, including, for example, antibiotics (e.g., penicillinand tetracycline); enzymes; vitamins (e.g., riboflavin, B12,beta-carotene); and hormones. In a preferred embodiment the (desired)fermentation product is ethanol.

According to the invention the desired fermentation product may berecovered by distillation. According to the invention oil may berecovered from the thin stillage and/or syrup/evaporated centrate, e.g.,by extraction, such as hexane extraction.

In a specific embodiment the process of the invention relates torecovering oil comprising

-   -   (a) converting a starch-containing material into dextrins with        an alpha-amylase;    -   (b) saccharifying the dextrins using a carbohydrate source        generating enzyme to form a sugar;    -   (c) fermenting the sugar in a fermentation medium into a        fermentation product using a fermenting organism;    -   (d) recovering the fermentation product to form a whole        stillage;    -   (e) separating the whole stillage into thin stillage and wet        cake;    -   (e′) concentrating the thin stillage into syrup;    -   (f) recovering oil from the syrup, wherein a protease having a        thermostability value of more than 20% determined as Relative        Activity at 80° C./70° C. is present and/or added in step (e′).

In another specific embodiment the process of recovering oil comprises

-   -   (a) converting a starch-containing material into dextrins with        an alpha-amylase;    -   (b) saccharifying the dextrins using a carbohydrate source        generating enzyme to form a sugar;    -   (c) fermenting the sugar in a fermentation medium into a        fermentation product using a fermenting organism;    -   (d) optionally recovering the fermentation product to form a        whole stillage;    -   (e) optimally separating the whole stillage into thin stillage        and wet cake;    -   (e′) optionally concentrating the thin stillage into syrup;        wherein a protease having a thermostability value of more than        20% determined as Relative Activity at 80° C./70° C. is present        and/or added in step (a) and/or steps (d)-(e′) and oil is        recovered during and/or after step (a).

In another specific embodiment the process of recovering oil comprises

-   -   (a) converting a starch-containing material into dextrins with        an alpha-amylase;    -   (b) saccharifying the dextrins using a carbohydrate source        generating enzyme to form a sugar;    -   (c) fermenting the sugar in a fermentation medium into a        fermentation product using a fermenting organism;    -   (d) recovering the fermentation product;        wherein a protease having a thermostability value of more than        20% determined as Relative Activity at 80° C./70° C. is present        and/or added in step (a) and oil is recovered during and/or        after step (a).

In another specific embodiment the process of recovering oil comprises

-   -   (a) converting a starch-containing material into dextrins with        an alpha-amylase and Pyrococcus furiosus protease;    -   (b) saccharifying the dextrins using a carbohydrate source        generating enzyme to form a sugar;    -   (c) fermenting the sugar in a fermentation medium into a        fermentation product using a fermenting organism;    -   (d) recovering the fermentation product, wherein oil is        recovered during and/or after step (a).        After step (a) means after step (a) and before saccharification        in step (b).

In a preferred embodiment the protease is derived from a strain ofPyrococcus, preferably a strain of Pyrococcus furiosus, such as the oneshown in SEQ ID NO: 1 in U.S. Pat. No. 6,258,726 or SEQ ID NO: 4 herein.In an embodiment the protease has at least 60%, such as at least 70%,such as at least 80%, such as at least 85%, such as at least 90%, suchas at least 95%, such as at least 96%, such as at least 97%, such as atleast 98%, such as at least 99% sequence identity to SEQ ID NO: 1 inU.S. Pat. No. 6,258,726 or SEQ ID NO: 4 herein. Other contemplatedproteases are described in the “Proteases” section below.

Use of Thermostable Protease for Improving Oil Extraction

In an aspect, the invention relates to the use of a protease having athermostability value of more than 20%, more than 30%, more than 40%,more than 50%, more than 60%, more than 70%, more than 80%, more than90% more than 100%, such as more that 105%, such as more than 110%, suchas more than 115%, such as more than 120% determined as RelativeActivity at 80° C./70° C. for increasing oil recovery yields from thinstillage and/or syrup in a fermentation product production process. Inan embodiment the protease may have a thermostability of above 90%,above 100% at 85° C. as determined using the Zein-BCA assay as disclosedin Example 2.Separating (Dewatering) Whole Stillage into Thin Stillage and Wet Cakein Step (e)

Separating whole stillage into thin stillage and wet cake in step (e),in order to remove a significant portion of the liquid/water, may bedone using any suitable separation technique, including centrifugation,pressing and filtration. In a preferred embodiment theseparation/dewatering is carried out by centrifugation. Preferredcentrifuges in industry are decanter type centrifuges, preferably highspeed decanter type centrifuges. An example of a suitable centrifuge isthe NX 400 steep cone series from Alfa Laval which is a high-performancedecanter. In another preferred embodiment the separation is carried outusing other conventional separation equipment such as a plate/framefilter presses, belt filter presses, screw presses, gravity thickenersand deckers, or similar equipment.

Drying of Wet Cake

After the wet cake, containing about 30-35 wt-% dry solids, has beenseparated from the thin stillage (e.g., dewatered) it may be dried in adrum dryer, spray dryer, ring drier, fluid bed drier or the like inorder to produce “Distillers Dried Grains” (DDG). DDG is a valuable feedingredient for livestock, poultry and fish. It is preferred to provideDDG with a content of less than about 10-12 wt.-% moisture to avoid moldand microbial breakdown and increase the shelf life. Further, highmoisture content also makes it more expensive to transport DDG. The wetcake is preferably dried under conditions that do not denature proteinsin the wet cake. The wet cake may be blended with syrup separated fromthe thin stillage and dried into DDG with Solubles (DDGS).

Fermenting Organisms

Examples of fermenting organisms used in step c) for fermenting sugarsin a fermentation medium into a desired fermentation product includefungal organisms, such as especially yeast. Preferred yeast includesstrains of Saccharomyces spp., in particular, Saccharomyces cerevisiae.

In one embodiment the fermenting organism is added to the fermentationmedium, so that the viable fermenting organism, such as yeast, count permL of fermentation medium is in the range from 10⁵ to 10¹², preferablyfrom 10⁷ to 10¹⁰, especially about 5×10⁷.

Commercially available yeast includes, e.g., RED STAR™ and ETHANOL RED□yeast (available from Fermentis/Lesaffre, USA), FALI (available fromFleischmann's Yeast, USA), SUPERSTART and THERMOSACC™ fresh yeast(available from Ethanol Technology, WI, USA), BIOFERM AFT and XR(available from NABC—North American Bioproducts Corporation, GA, USA),GERT STRAND (available from Gert Strand AB, Sweden), and FERMIOL(available from DSM Specialties).

Starch-Containing Materials

Any suitable starch-containing material may be used according to thepresent invention. The starting material is generally selected based onthe desired fermentation product. Examples of starch-containingmaterials, suitable for use in a process of the invention, include wholegrains, corn, wheat, barley, rye, milo, sago, cassava, tapioca, sorghum,rice, peas, beans, or sweet potatoes, or mixtures thereof or starchesderived there from, or cereals. Contemplated are also waxy and non-waxytypes of corn and barley.

Fermentation Products

The term “fermentation product” means a product produced by a processincluding a fermentation step using a fermenting organism. Fermentationproducts contemplated according to the invention include alcohols (e.g.,ethanol, methanol, butanol); organic acids (e.g., citric acid, aceticacid, itaconic acid, lactic acid, succinic acid, gluconic acid); ketones(e.g., acetone); amino acids (e.g., glutamic acid); gases (e.g., H2 andCO2); antibiotics (e.g., penicillin and tetracycline); enzymes; vitamins(e.g., riboflavin, B12, beta-carotene); and hormones. In a preferredembodiment the fermentation product is ethanol, e.g., fuel ethanol;drinking ethanol, i.e., potable neutral spirits; or industrial ethanolor products used in the consumable alcohol industry (e.g., beer andwine), dairy industry (e.g., fermented dairy products), leather industryand tobacco industry. Preferred beer types comprise ales, stouts,porters, lagers, bitters, malt liquors, happoushu, highalcohol beer,low-alcohol beer, low-calorie beer or light beer. Preferred fermentationprocesses used include alcohol fermentation processes. The fermentationproduct, such as ethanol, obtained according to the invention, maypreferably be used as fuel, that typically is blended with gasoline.However, in the case of ethanol it may also be used as potable ethanol.

Recovery

Subsequent to fermentation the fermentation product, such as ethanol maybe separated from the fermentation medium, e.g., by distillation.Alternatively the desired fermentation product may be extracted from thefermentation medium by micro or membrane filtration techniques. Thefermentation product may also be recovered by stripping or other methodwell known in the art.

Enzymes

One or more of the following enzyme activities may be used according tothe invention.

Proteases

According to the invention a thermostable protease (as defined herein)may be present of added during step (a) or steps (d)-(e′).

The protease may be of any origin as long as it has a thermostabilityvalue as defined herein of more than 20% and the Example 1. In anembodiment the protease is of fungal origin. In another embodiment theprotease is of bacterial origin. In an embodiment protease has athermostability of more than 30%, more than 40%, more than 50%, morethan 60%, more than 70%, more than 80%, more than 90% more than 100%,such as more that 105%, such as more than 110%, such as more than 115%,such as more than 120% determined as Relative Activity at 80° C./70° C.

In another embodiment the protease has a thermostability value between50 and 115%, such as between 50 and 70%, such as between 50 and 60%,such as between 100 and 120%, such as between 105 and 115% determined asRelative Activity at 80° C./70° C.

In an embodiment the protease has a thermostability value of more than12%, more than 14%, more than 16%, more than 18%, more than 20%, morethan 25%, more than 30%, more than 40%, more that 50%, more than 60%,more than 70%, more than 80%, more than 90%, more than 100%, more than110% determined as Relative Activity at 85° C./70° C.

In an embodiment the protease has a thermostability value between 10 and50%, such as between 10 and 30%, such as between 10 and 25% determinedas Relative Activity at 85° C./70° C.

In an embodiment the protease has a thermostability value between 50 and110%, such as between 70 and 110%, such as between 90 and 110%determined as Relative Activity at 85° C./70° C. In an embodiment theprotease may have a thermostability of above 90%, above 100% at 85° C.as determined using the Zein-BCA assay as disclosed in Example 2.

Fungal Proteases

In an embodiment the protease is of fungal origin.

In a preferred embodiment the protease is a variant of the metalloprotease derived from a strain of the genus Thermoascus, preferably astrain of Thermoascus aurantiacus, especially Thermoascus aurantiacusCGMCC No. 0670.

In an embodiment the protease is a variant of the metallo proteasedisclosed as the mature part of SEQ ID NO: 2 disclosed in WO 2003/048353or SEQ ID NO: 3 herein.

In an embodiment the parent protease has at least 70%, such as at least75% identity preferably at least 80%, more preferably at least 85%, morepreferably at least 90%, more preferably at least 91%, more preferablyat least 92%, even more preferably at least 93%, most preferably atleast 94%, and even most preferably at least 95%, such as even at least96%, at least 97%, at least 98%, at least 99%, such as least 100%identity to the mature part of the polypeptide of SEQ ID NO: 2 disclosedin WO 2003/048353 or SEQ ID NO: 3 herein.

In an embodiment the protease variant has at least 70%, such as at least75% identity preferably at least 80%, more preferably at least 85%, morepreferably at least 90%, more preferably at least 91%, more preferablyat least 92%, even more preferably at least 93%, most preferably atleast 94%, and even most preferably at least 95%, such as even at least96%, at least 97%, at least 98%, at least 99%, but less than 100%identity to the mature part of the polypeptide of SEQ ID NO: 2 disclosedin WO 2003/048353 or SEQ ID NO: 3 herein.

In an embodiment the protease is a variant shown in any of Tables 1-4 inExamples 1 or 2 having a thermostability value of more than 20%, morethan 30%, more than 40%, more than 50%, more than 60%, more than 70%,more than 80%, more than 90% more than 100%, such as more that 10⁵%,such as more than 110%, such as more than 115%, such as more than 120%determined as Relative Activity at 80° C./70° C.

This includes the protease variants of the wild-type protease shown inSEQ ID NO: 3, having the following substitutions:

-   -   D79L+S87P+A112P+D142L    -   D79L+Y82F+S87P+A112P+D142L    -   S38T+D79L+S87P+A112P+A126V+D142L    -   D79L+Y82F+S87P+A112P+A126V+D142L    -   A27K+D79L+S87P+A112P+A126V+D142L    -   S49P+D79L+S87P+A112P+D142L    -   S50P+D79L+S87P+A112P+D142L    -   D79L+S87P+D104P+A112P+D142L    -   D79L+Y82F+S87G+A112P+D142L    -   S70V+D79L+Y82F+S87G+Y97W+A112P+D142L    -   D79L+Y82F+S87G+Y97W+D104P+A112P+D142L    -   S70V+D79L+Y82F+S87G+A112P+D142L    -   D79L+Y82F+S87G+D104P+A112P+D142L    -   D79L+Y82F+S87G+A112P+A126V+D142L    -   Y82F+S87G+S70V+D79L+D104P+A112P+D142L    -   Y82F+S87G+D79L+D104P+A112P+A126V+D142L    -   A27K+D79L+Y82F+S87G+D104P+A112P+A126V+D142L    -   A27K Y82F S87G D104P A112P A126V D142L    -   A27K D79L Y82F D104P A112P A126V D142L    -   A27K Y82F D104P A112P A126V D142L

In a preferred embodiment the protease is a variant of the Thermoascusaurantiacus protease shown in SEQ ID NO: 3 herein with mutationsselected from the group consisting of:

-   -   A27K+D79L+Y82F+S87G+D104P+A112P+A126V+D142L;    -   D79L+Y82F+S87G+A112P+D142L;    -   Y82F+S87G+S70V+D79L+D104P+A112P+D142L;    -   Y82F+S87G+D79L+D104P+A112P+A126V+D142L.

In an additional embodiment the protease is a filamentous fungus, e.g.,derived from a strain of Rhizomucor, such as Rhizomucor miehei, such asthe protease shown in SEQ ID NO: 9 or one having a sequence identitythereto of at least 60%, at least 70%, at least 80%, at least 90%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%.

Bacterial Proteases

In an embodiment the protease is of bacterial origin.

In a preferred embodiment the protease is derived from a strain ofPyrococcus, preferably a strain of Pyrococcus furiosus.

In a preferred embodiment the protease is the one shown in SEQ ID NO: 1in U.S. Pat. No. 6,258,726 or SEQ ID NO: 4 herein.

In an embodiment the protease has at least 60%, such as at least 70%,such as at least 80%, such as at least 85%, such as at least 90%, suchas at least 95%, such as at least 96%, such as at least 97%, such as atleast 98%, such as at least 99% sequence identity to SEQ ID NO: 1 inU.S. Pat. No. 6,258,726 or SEQ ID NO: 4 herein.

In an embodiment the protease is present and/or added in an effectiveamount in step (a) or any of steps (d)-(e′). In an embodiment of theinvention the protease is added in a concentration of 0.01-100, such0.1-10 micro g/g DS.

Alpha-Amylases

The method of the invention, including step (a), may be carried outusing any suitable alpha-amylase. In a preferably embodiment a bacterialalpha-amylase and/or a fungal alpha-amylase may be used.

In an embodiment the alpha-amylase is bacterial when step (a) is carriedout as a liquefaction step at high temperatures, i.e., above the initialgelatinization temperature;

In an embodiment the alpha-amylase is fungal when step (a) is carriedout at a temperature below the initial gelatinization temperature, suchas when steps (a), (b) and (c) are carried out as a raw starchhydrolysis (single step process or no-cook process) as described above.

Bacterial Alpha-Amylases

Examples of suitable bacterial alpha-amylases include the belowmentioned. Preferred bacterial alpha-amylases used in step i) may bederived from a strain the genus Bacillus (sometimes referred to asGeobacillus), including a strain of Bacillus licheniformis, Bacillusamyloliquefaciens, Bacillus stearothermophilus, or Bacillus subtilis.Other bacterial alpha-amylases include alpha-amylase derived from astrain of the Bacillus sp. NCIB 12289, NCIB 12512, NCIB 12513 or DSM9375, all of which are described in detail in WO 95/26397, and thealpha-amylase described by Tsukamoto et al., Biochemical and BiophysicalResearch Communications, 151 (1988), pp. 25-31 (hereby incorporated byreference).

The Bacillus alpha-amylase may also be a variant and/or hybrid,especially one described in any of WO 96/23873, WO 96/23874, WO97/41213, WO 99/19467, WO 00/60059, and WO 02/10355 (all documentshereby incorporated by reference). Specifically contemplatedalpha-amylase variants are disclosed in U.S. Pat. Nos. 6,093,562,6,297,038 or U.S. Pat. No. 6,187,576 (hereby incorporated by reference)and include Bacillus stearothermophilus alpha-amylase (BSGalpha-amylase) variants having a deletion of one or two amino acid inpositions R179 to G182, preferably a double deletion disclosed in WO1996/023873—see e.g., page 20, lines 1-10 (hereby incorporated byreference), preferably corresponding to delta(181-182) compared to thewild-type BSG alpha-amylase amino acid sequence set forth in SEQ ID NO:3disclosed in WO 99/19467 or deletion of amino acids R179 and G180 usingSEQ ID NO:3 in WO 99/19467 for numbering (which reference is herebyincorporated by reference). Even more preferred are Bacillusalpha-amylases, especially Bacillus stearothermophilus alpha-amylase,which have a double deletion corresponding to delta(181-182) and furthercomprise a N193F substitution (also denoted I181*+G182*+N193F) comparedto the wild-type BSG alpha-amylase amino acid sequence set forth in SEQID NO:3 disclosed in WO 99/19467.

In an embodiment the Bacillus stearothermophilus alpha-amylase is onedisclosed in WO 2011/082425, such as one selected from the group of:

-   -   I181*+G182*+N193F+E129V+K177L+R179E;    -   I181*+G182*+N193F+V59A+Q89R+E129V+K177L+R179E+H208Y+K220P+N224L+Q254S;    -   I181*+G182*+N193F+V59A+Q89R+E129V+K177L+R179E+Q254S+M284V; and    -   I181*+G182*+N193F+E129V+K177L+R179E+K220P+N224L+S242Q+Q254S        (using SEQ ID NO: 1 herein for numbering).

In an embodiment the Bacillus stearothermophilus alpha-amylase has thefollowing mutations:181*+G182*+N193F+V59A+Q89R+E129V+K177L+R179E+Q254S+M284V (SEQ ID NO: 1).

The truncated Bacillus stearothermophilus alpha-amylase are typicallynaturally truncated to be about 491 amino acids long, such as from485-495 amini acids long.

A hybrid alpha-amylase specifically contemplated comprises 445C-terminal amino acid residues of the Bacillus licheniformisalpha-amylase (shown in SEQ ID NO: 4 of WO 99/19467) and the 37N-terminal amino acid residues of the alpha-amylase derived fromBacillus amyloliquefaciens (shown in SEQ ID NO: 5 of WO 99/19467), withthe following substitution:G48A+T491+G10⁷A+H156Y+A181T+N190F+1201F+A209V+Q264S (using the numberingin SEQ ID NO: 4 in WO 99/19467). Especially preferred are variantshaving one or more of the mutations H154Y, A181T, N190F, A209V and Q264Sand/or deletion of two residues between positions 176 and 179,preferably deletion of E178 and G179 (using the SEQ ID NO: 5 numberingof WO 99/19467).

Commercially available bacterial alpha-amylase products and productscontaining alphaamylases include TERMAMYL™ SC, LIQUOZYME™ SC, BAN(Novozymes A/S, Denmark) DEX-LO™, SPEZYME™ XTRA, SPEZYME™ AA, SPEZYMEFRED-L, SPEZYME™ ALPHA, GC358, SPEZYME RSL, SPEZYME HPA and SPEZYME™DELTA AA (from DuPont, USA), FUELZYME™ (Verenium, USA).

A bacterial alpha-amylase may be added in step (a) in amounts as arewell-known in the art. When measured in KNU units (described below inthe “Materials & Methods”-section) the alpha-amylase activity ispreferably present in an amount of 0.5-5,000 NU/g of DS, in an amount of1-500 NU/g of DS, or more preferably in an amount of 5-1,000 NU/g of DS,such as 10-100 NU/g DS.

Fungal Alpha-Amylases

Fungal alpha-amylases (EC 3.2.1.1) are preferably of filamentous fungusorigin. The fungal alpha-amylase may be a fungal acid alpha-amylase.

Fungal acid alpha-amylases include acid alpha-amylases derived from astrain of the genus Aspergillus, such as Aspergillus oryzae andAspergillus niger alpha-amylases.

A preferred fungal alpha-amylase is a Fungamyl-like alpha-amylase whichis preferably derived from a strain of Aspergillus oryzae. In thepresent disclosure, the term “Fungamyl-like alpha-amylase” indicates analpha-amylase which exhibits a high identity, i.e. more than 70%, morethan 75%, more than 80%, more than 85% more than 90%, more than 95%,more than 96%, more than 97%, more than 98%, more than 99% or even 100%identity to the mature part of the amino acid sequence shown in SEQ IDNO: 10 in WO 96/23874.

Another preferred acid alpha-amylase is derived from a strainAspergillus niger. In a preferred embodiment the acid fungalalpha-amylase is the one from A. niger disclosed as “AMYA_ASPNG” in theSwiss-prot/TeEMBL database under the primary accession no. P56271 anddescribed in more detail in WO 89/01969 (Example 3). The acidAspergillus niger acid alpha-amylase is also shown as SEQ ID NO: 1 in WO2004/080923 (Novozymes) which is hereby incorporated by reference. Alsovariants of said acid fungal amylase having at least 70% identity, suchas at least 80% or even at least 90% identity, such as at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% identity to SEQID NO: 1 in WO 2004/080923 are contemplated. A suitable commerciallyavailable acid fungal alpha-amylase derived from Aspergillus niger isSP288 (available from Novozymes A/S, Denmark).

The fungal acid alpha-amylase may also be a wild-type enzyme comprisinga carbohydrate-binding module (CBM) and an alpha-amylase catalyticdomain (i.e., a none-hybrid), or a variant thereof. In an embodiment thewild-type acid fungal alpha-amylase is derived from a strain ofAspergillus kawachii.

Commercial available compositions comprising fungal alpha-amylaseinclude FUNGAMYL™ and the acid fungal alpha-amylase sold under the tradename SP288 (available from Novozymes A/S, Denmark).

In an embodiment the fungal acid alpha-amylase is a hybridalpha-amylase. Preferred examples of fungal hybrid alpha-amylasesinclude the ones disclosed in WO 2005/003311 or U.S. Patent Publicationno. 2005/0054071 (Novozymes) or U.S. patent application No. 60/638,614(Novozymes) which is hereby incorporated by reference. A hybridalpha-amylase may comprise an alpha-amylase catalytic domain (CD) and acarbohydrate-binding domain/module (CBM), such as a starch bindingdomain, and optional a linker.

Specific examples of contemplated hybrid alpha-amylases include thosedisclosed in Table 1 to 5 of the examples in co-pending U.S. patentapplication No. 60/638,614, including Fungamyl variant with catalyticdomain JA118 and Athelia rolfsii SBD (SEQ ID NO: 2 herein and SEQ IDNO:100 in U.S. 60/638,614), Rhizomucor pusillus alpha-amylase withAthelia rolfsii AMG linker and SBD (SEQ ID NO: 3 herein and SEQ IDNO:101 in U.S. 60/638,614), Rhizomucor pusillus alpha-amylase withAspergillus niger glucoamylase linker and SBD (which is disclosed inTable 5 as a combination of amino acid sequences SEQ ID NO:20 SEQ IDNO:72 and SEQ ID NO:96 in U.S. application Ser. No. 11/316,535 andfurther as SEQ ID NO: 13 herein), and Meripilus giganteus alpha-amylasewith Athelia rolfsii glucoamylase linker and SBD (SEQ ID NO: 4 hereinand SEQ ID NO:102 in U.S. 60/638,614). Other specifically contemplatedhybrid alpha-amylases are any of the ones listed in Tables 3, 4, 5, and6 in Example 4 in U.S. application Ser. No. 11/316,535 or (WO2006/069290) (hereby incorporated by reference). Other specific examplesof contemplated hybrid alpha-amylases include those disclosed in U.S.Patent Publication no. 2005/0054071, including those disclosed in Table3 on page 15, such as Aspergillus niger alpha-amylase with Aspergilluskawachii linker and starch binding domain.

An acid alpha-amylases may according to the invention be added in anamount of 0.1 to 10 AFAU/g DS, preferably 0.10 to 5 AFAU/g DS,especially 0.3 to 2 AFAU/g DS.

Fungal alpha-amylases may be added to step (a) in a well know effectiveamount, preferably in the range from 0.001-1 mg enzyme protein per g DS(in whole stillage), preferably 0.01-0.5 mg enzyme protein per g DS.

Carbohydrate-Source Generating Enzyme

According to the invention a carbohydrate-source generating enzyme,preferably a glucoamylase, may be present and/or added duringsaccharification step (b) or simultaneous saccharification andfermentation.

The term “carbohydrate-source generating enzyme” includes any enzymesgenerating fermentable sugars. A carbohydrate-source generating enzymeis capable of producing a carbohydrate that can be used as anenergy-source by the fermenting organism(s) in question, for instance,when used in a process of the invention for producing a fermentationproduct, such as ethanol. The generated carbohydrates may be converteddirectly or indirectly to the desired fermentation product, preferablyethanol. According to the invention a mixture of carbohydrate-sourcegenerating enzymes may be used. Specific examples include glucoamylase(being glucose generators), beta-amylase and maltogenic amylase (beingmaltose generators).

In a preferred embodiment the carbohydrate-source generating enzyme is aglucoamylase.

Glucoamylases

The process of the invention, including step (b), may be carried outusing any suitable glucoamylase. In a preferably embodiment theglucoamylase is of bacterial or fungal origin.

Contemplated glucoamylases include those from the group consisting ofAspergillus glucoamylases, in particular A. niger G1 or G2 glucoamylase(Boel et al. (1984), EMBO J. 3 (5), p. 1097-1102), or variants thereof,such as those disclosed in WO 92/00381, WO 00/04136 and WO 01/04273(from Novozymes, Denmark); the A. awamori glucoamylase disclosed in WO84/02921, A. oryzae glucoamylase (Agric. Biol. Chem. (1991), 55 (4), p.941-949), or variants or fragments thereof. Other Aspergillusglucoamylase variants include variants with enhanced thermal stability:G137A and G139A (Chen et al. (1996), Prot. Eng. 9, 499-505); D257E andD293E/Q (Chen et al. (1995), Prot. Eng. 8, 575-582); N182 (Chen et al.(1994), Biochem. J. 301, 275-281); disulphide bonds, A246C (Fierobe etal. (1996), Biochemistry, 35, 8698-8704; and introduction of Proresidues in position A435 and S436 (Li et al. (1997), Protein Eng. 10,1199-1204.

Other glucoamylases contemplated include glucoamylase derived from astrain of Athelia, preferably a strain of Athelia rolfsii (previouslydenoted Corticium rolfsii) glucoamylase (see U.S. Pat. No. 4,727,026 and(Nagasaka, Y. et al. (1998) “Purification and properties of theraw-starch-degrading glucoamylases from Corticium rolfsii, ApplMicrobiol Biotechnol 50:323-330), Talaromyces glucoamylases, inparticular derived from Talaromyces emersonii (WO 99/28448), Talaromycesleycettanus (US patent no. Re. 32,153), Talaromyces duponti, Talaromycesthermophilus (U.S. Pat. No. 4,587,215). Also contemplated are theTrichoderma reesei glucoamylases disclosed as SEQ ID NO: 4 in WO2006/060062 and glucoamylases being at least 80% or at least 90%identical thereto and further the glucoamylase derived from Humicolagrisea disclosed as SEQ ID NO: 3 in U.S. Ser. No. 10/992,187 (herebyincorporated by reference) or sequences having at least 80% or at least90% identity thereto.

In a preferred embodiment the glucoamylase is derived from a strain ofAspergillus, preferably A. niger, A. awamori, or A. oryzae; or a strainof Trichoderma, preferably T. reesei; or a strain of Talaromyces,preferably T. emersonii.

In an embodiment the glucoamylase present and/or added duringsaccharification and/or fermentation is of fungal origin, preferablyfrom a strain of Pycnoporus, or a strain of Gloephyllum.

In an embodiment the glucoamylase is derived from a strain of the genusPycnoporus, in particular a strain of Pycnoporus sanguineus described inWO 2011/066576 (SEQ ID NOs 2, 4 or 6), such as the one shown as SEQ IDNO: 4 in WO 2011/066576 or SEQ ID NO: 18 herein.

In an embodiment the glucoamylase is derived from a strain of the genusGloeophyllum, such as a strain of Gloeophyllum sepiarium or Gloeophyllumtrabeum, in particular a strain of Gloeophyllum as described in WO2011/068803 (SEQ ID NO: 2, 4, 6, 8, 10, 12, 14 or 16). In a preferredembodiment the glucoamylase is the Gloeophyllum sepiarium shown in SEQID NO: 2 in WO 2011/068803 or SEQ ID NO: 15 herein.

Other contemplated glucoamylases include glucoamylase derived from astrain of Trametes, preferably a strain of Trametes cingulata disclosedin WO 2006/069289 (which is hereby incorporated by reference). Alsohybrid glucoamylase are contemplated according to the invention.Examples the hybrid glucoamylases disclosed in WO 2005/045018. Specificexamples include the hybrid glucoamylase disclosed in Table 1 and 4 ofExample 1 (which hybrids are hereby incorporated by reference).

Bacterial glucoamylases contemplated include glucoamylases from thegenus Clostridium, in particular C. thermoamylolyticum (EP 135,138), andC. thermohydrosulfuricum (WO 86/01831).

Commercially available compositions comprising glucoamylase include AMG200L; AMG 300 L; SAN™ SUPER, SAN™ EXTRA L, SPIRIZYME™ PLUS, SPIRIZYME™FUEL, SPIRIZYME™ B4U and AMG™ E (from Novozymes A/S); OPTIDEX™ 300 (fromGenencor Int.); AMIGASE™ and AMIGASE™ PLUS (from DSM); G-ZYME™ G900,G-ZYME™ and G990 ZR (from Genencor Int.).

Glucoamylases may in an embodiment be added in an amount of 0.02-20AGU/g DS, preferably 0.05-5 AGU/g DS (in whole stillage), especiallybetween 0.1-2 AGU/g DS.

Glucoamylase may be added in an effective amount, preferably in therange from 0.001-1 mg enzyme protein per g DS, preferably 0.01-0.5 mgenzyme protein per g dry substrate.

The invention described and claimed herein is not to be limited in scopeby the specific embodiments herein disclosed, since these embodimentsare intended as illustrations of several aspects of the invention. Anyequivalent embodiments are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims. In the case ofconflict, the present disclosure including definitions will control.Various references are cited herein, the disclosures of which areincorporated by reference in their entireties. The present invention isfurther described by the following examples which should not beconstrued as limiting the scope of the invention.

Material & Methods Enzymes:

Alpha-Amylase LSCDS (“LSCDS”): Bacillus stearothermophilus alpha-amylasewith the mutations: I181*+G182*+N193F truncated to 491 amino acids (SEQID NO: 1 herein).Alpha-Amylase 369: (AA369): Bacillus stearothermophilus alpha-amylasewith the mutations:1181*+G182*+N193F+V59A+Q89R+E129V+K177L+R179E+Q254S+M284V truncated to491 amino acids (SEQ ID NO: 1 herein).Protease TA (“TA”): Metallo protease derived from Thermoascusaurantiacus CGMCC No. 0670 disclosed as amino acids 1-177 in SEQ ID NO:3 herein Protease 196 (“TA 196”): Metallo protease derived fromThermoascus aurantiacus CGMCC No. 0670 disclosed as amino acids 1-177 inSEQ ID NO: 3 herein with the following mutations:A27K+D79L+Y82F+S87G+D104P+A112P+A126V+D142L.Protease PF (“PF”): Protease derived from the bacterium Pyrococcusfuriosus shown in SEQ ID NO: 4 herein.Protease RH (“RH”): Protease derived from a filamentous fungusRhizomucor miehei shown in SEQ ID NO: 9 herein.Protease TF (“TF”): Protease derived from a filamentous fungusThermobifida fusca shown in SEQ ID NO: 10 herein.Glucoamylase SF is a glucoamylase derived from a strain of Talaromycesemersonii and is disclosed in WO9928448 and is available from NovozymesA/S.Glucoamylase TC is a glucoamylase derived from Trametes cingulatadisclosed in SEQ ID NO: 2 of WO 2006/069289 and available from NovozymesA/S.Alpha-amylase JA is an alpha-amylase derived from Rhizomucor pusillusand disclosed as V039 in Table 5 in WO 2006/069290.

Determination of Alpha-Amylase Activity 1. Phadebas™ Assay

Alpha-amylase activity is determined by a method employing Phadebas®tablets as substrate. Phadebas tablets (Phadebas® Amylase Test, suppliedby Pharmacia Diagnostic) contain a cross-linked insoluble blue-coloredstarch polymer, which has been mixed with bovine serum albumin and abuffer substance and tableted.

For every single measurement one tablet is suspended in a tubecontaining 5 ml 50 mM Britton-Robinson buffer (50 mM acetic acid, 50 mMphosphoric acid, 50 mM boric acid, 0.1 mM CaCl₂), pH adjusted to thevalue of interest with NaOH). The test is performed in a water bath atthe temperature of interest. The alpha-amylase to be tested is dilutedin x ml of 50 mM Britton-Robinson buffer. 1 ml of this alpha-amylasesolution is added to the 5 ml 50 mM Britton-Robinson buffer. The starchis hydrolyzed by the alpha-amylase giving soluble blue fragments. Theabsorbance of the resulting blue solution, measuredspectrophotometrically at 620 nm, is a function of the alpha-amylaseactivity.

It is important that the measured 620 nm absorbance after 10 or 15minutes of incubation (testing time) is in the range of 0.2 to 2.0absorbance units. In this absorbance range there is linearity betweenactivity and absorbance (Lambert-Beer law). The dilution of the enzymemust therefore be adjusted to fit this criterion. Under a specified setof conditions (temperature, pH, reaction time, buffer conditions) 1 mgof a given alpha-amylase will hydrolyze a certain amount of substrateand a blue colour will be produced. The measured absorbance is directlyproportional to the specific activity (activity/mg of pure alpha-amylaseprotein) of the alpha-amylase in question under the given set ofconditions.

2. Alternative Method

Alpha-amylase activity is alternatively determined by a method employingthe PNP-G7 substrate. PNP-G7 which is a abbreviation forp-nitrophenyl-alpha,D-maltoheptaoside is a blocked oligosaccharide whichcan be cleaved by an endo-amylase. Following the cleavage, thealpha-glucosidase included in the kit digest the substrate to liberate afree PNP molecule which has a yellow colour and thus can be measured byvisible spectophometry at wavelength Lambda=405 nm (400-420 nm). Kitscontaining PNP-G7 substrate and alpha-glucosidase is manufactured byBohringer-Mannheim (cat. No. 1054635).

To prepare the substrate one bottle of substrate (BM 1442309) is addedto 5 ml buffer (BM1442309). To prepare the alpha-glucosidase one bottleof alpha-glucosidase (BM 1462309) is added to 45 ml buffer (BM1442309).The working solution is made by mixing 5 ml alpha-glucosidase solutionwith 0.5 ml substrate.

The assay is performed by transforming 20 microL enzyme solution to a 96well microtitre plate and incubating at 25° C. 200 microL workingsolution, 25° C. is added. The solution is mixed and pre-incubated 1minute and absorption is measured every 15 seconds over 3 minutes at OD405 nm.

The slope of the time dependent absorption-curve is directlyproportional to the specific activity (activity per mg enzyme) of thealpha-amylase in question under the given set of conditions.

Determination of Acid Amylolytic Activity (FAU)

One Fungal Alpha-Amylase Unit (1 FAU) is defined as the amount ofenzyme, which breaks down 5.26 g starch (Merck Amylum solubile Erg. B.6,Batch 9947275) per hour at Novozymes' standard method for determinationof alpha-amylase based upon the following standard conditions:

Substrate Soluble starch Temperature 37° C. pH 4.7 Reaction time 7-20minutesA detailed description of Novozymes' method for determining KNU and FAUis available on request as standard method EB-SM-0009.02/01.

Determination of Acid Alpha-Amylase Activity (AFAU)

Acid alpha-amylase activity is measured in AFAU (Acid FungalAlpha-amylase Units), which are determined relative to an enzymestandard.

The standard used is AMG 300 L (wild type A. niger G1 AMG sold byNovozymes A/S). The neutral alpha-amylase in this AMG falls afterstorage at room temperature for 3 weeks from approx. 1 FAU/mL to below0.05 FAU/mL.

The acid alpha-amylase activity in this AMG standard is determined inaccordance with AF 9⅓ (Novo method for the determination of fungalalpha-amylase). In this method, 1 AFAU is defined as the amount ofenzyme, which degrades 5.260 mg starch dry matter per hour understandard conditions.

Iodine forms a blue complex with starch but not with its degradationproducts. The intensity of colour is therefore directly proportional tothe concentration of starch. Amylase activity is determined usingreverse colorimetry as a reduction in the concentration of starch underspecified analytic conditions.

Blue/violet t=23 sec. DecolourationStandard conditions/reaction conditions: (per minute)Substrate: starch, approx. 0.17 g/LBuffer: Citrate, approx. 0.03 M

Iodine (I₂): 0.03 g/L CaCl₂: 1.85 mM

pH: 2.50±0.05Incubation temperature: 40° C.Reaction time: 23 seconds

Wavelength: Lambda=590 nm

Enzyme concentration: 0.025 AFAU/mLEnzyme working range: 0.01-0.04 AFAU/mL

Further details can be found in standard method documentEB-SM-0259.02/01 available on request from Novozymes A/S, which folderis hereby incorporated by reference.

Determination of FAU-F

FAU-F Fungal Alpha-Amylase Units (Fungamyl) is measured relative to anenzyme standard of a declared strength.

Reaction conditions Temperature 37° C. pH 7.15 Wavelength 405 nmReaction time   5 min Measuring time   2 min

A folder (EB-SM-0216.02) describing this standard method in more detailis available on request from Novozymes A/S, Denmark, which folder ishereby included by reference.

Alpha-amylase Activity (KNU)

The alpha-amylase activity may be determined using potato starch assubstrate. This method is based on the break-down of modified potatostarch by the enzyme, and the reaction is followed by mixing samples ofthe starch/enzyme solution with an iodine solution. Initially, ablackish-blue color is formed, but during the break-down of the starchthe blue color gets weaker and gradually turns into a reddish-brown,which is compared to a colored glass standard.One Kilo Novo alpha amylase Unit (KNU) is defined as the amount ofenzyme which, under standard conditions (i.e., at 37° C.+/−0.05; 0.0003M Ca²⁺; and pH 5.6) dextrinizes 5260 mg starch dry substance MerckAmylum solubile.A folder EB-SM-0009.02/01 describing this analytical method in moredetail is available upon request to Novozymes A/S, Denmark, which folderis hereby included by reference.

Glucoamylase and Alpha-Glucosidase Activity (AGU)

The Novo Glucoamylase Unit (AGU) is defined as the amount of enzyme,which hydrolyzes 1 micromole maltose per minute under the standardconditions 37° C., pH 4.3, substrate: maltose 23.2 mM, buffer: acetate0.1 M, reaction time 5 minutes.

An autoanalyzer system may be used. Mutarotase is added to the glucosedehydrogenase reagent so that any alpha-D-glucose present is turned intobeta-D-glucose. Glucose dehydrogenase reacts specifically withbeta-D-glucose in the reaction mentioned above, forming NADH which isdetermined using a photometer at 340 nm as a measure of the originalglucose concentration.

AMG Incubation:

Substrate: maltose 23.2 mM Buffer: acetate 0.1M pH: 4.30 ± 0.05Incubation temperature: 37° C. ± 1 Reaction time: 5 minutes Enzymeworking range: 0.5-4.0 AGU/mL

Color Reaction:

GlucDH: 430 U/L Mutarotase: 9 U/L NAD: 0.21 mM Buffer: phosphate 0.12M;0.15M NaCl pH: 7.60 ± 0.05 Incubation temperature: 37° C. ± 1 Reactiontime: 5 minutes Wavelength: 340 nm

A folder (EB-SM-0131.02/01) describing this analytical method in moredetail is available on request from Novozymes A/S, Denmark, which folderis hereby included by reference.

Determination of Protease Activity (AU)

Dimethyl casein (DMC) is hydrolyzed by the proteolytic enzyme to smallpeptides. The primary amino groups formed in this process react withtrinitrobenzene sul-phonic acid (TNBS) forming a coloured complex. Thiscolour development is monitored in situ so the change in absorption pertime unit can be calculated. This figure is a measure of the reactionrate and thus of the enzyme activity.

Reaction conditions for the DMC reaction Temperature: 50° C. pH: 8.3Wavelength: 405 nm Reaction time:   8 min. Measuring time:   2 min.Enzyme concentration range: 0.072-0.216 mAU/ml.The activity is determined relative to an enzyme standard.

The assay is further described in standard method documentEB-SM-0218.02/02 available upon request from Novozymes A/S, Denmark.

Relative Activity and Remaining Activity

“Relative Activity” and “Remaining Activity” is determined as describedin Example 1.

Themostability

Thermostability in Example 2 is determined using the Zein-BCA assay.

EXAMPLES Example 1 Preparation of Protease Variants and Test ofThermostability

Chemicals used were commercial products of at least reagent grade.

Strains and Plasmids:

E. coli DH12S (available from Gibco BRL) was used for yeast plasmidrescue. pJTP000 is a S. cerevisiae and E. coli shuttle vector under thecontrol of TPI promoter, constructed from pJC039 described in WO01/92502, in which the Thermoascus aurantiacus M35 protease gene (WO03/048353) has been inserted.

Saccharomyces cerevisiae YNG318 competent cells: MATa Dpep4[cir+]ura3-52, leu2-D2, his 4-539 was used for protease variants expression.It is described in J. Biol. Chem. 272(15): 9720-9727 (1997).

Media and Substrates

10× Basal solution: Yeast nitrogen base w/o amino acids (DIFCO) 66.8g/L, succinate 100 g/l, NaOH 60 g/l.SC-glucose: 20% glucose (i.e., a final concentration of 2%=2 g/100 mL))100 mL/L, 5% threonine 4 mL/L, 1% tryptophan10 ml/l, 20% casamino acids25 ml/l, 10× basal solution 100 ml/l. The solution is sterilized using afilter of a pore size of 0.20 micrometer. Agar (2%) and H₂O (approx. 761mL) is autoclaved together, and the separately sterilized SC-glucosesolution is added to the agar solution.YPD: Bacto peptone 20 g/l, yeast extract 10 g/L, 20% glucose 100 mL/L.

YPD+Zn: YPD+0.25 mM ZnSO₄.

PEG/LiAc solution: 40% PEG4000 50 ml, 5 M Lithium Acetate 1 mL.96 well Zein micro titre plate:

Each well contains 200 microL of 0.05-0.1% of zein (Sigma), 0.25 mMZnSO₄ and 1% of agar in 20 mM sodium acetate buffer, pH 4.5.

DNA Manipulations

Unless otherwise stated, DNA manipulations and transformations wereperformed using standard methods of molecular biology as described inSambrook et al. (1989) Molecular cloning: A laboratory manual, ColdSpring Harbor lab. Cold Spring Harbor, NY; Ausubel, F. M. et al. (eds.)“Current protocols in Molecular Biology”, John Wiley and Sons, 1995;Harwood, C. R. and Cutting, S. M. (Eds.).

Yeast Transformation

Yeast transformation was performed using the lithium acetate method. 0.5microL of vector (digested by restriction endnucleases) and 1 microL ofPCR fragments is mixed. The DNA mixture, 100 microL of YNG318 competentcells, and 10 microL of YEAST MAKER carrier DNA (Clontech) is added to a12 mL polypropylene tube (Falcon 2059). Add 0.6 mL PEG/LiAc solution andmix gently. Incubate for 30 min at 30° C., and 200 rpm followed by 30min at 42° C. (heat shock). Transfer to an eppendorf tube and centrifugefor 5 sec. Remove the supernatant and resolve in 3 mL of YPD. Incubatethe cell suspension for 45 min at 200 rpm at 30° C. Pour the suspensionto SC-glucose plates and incubate 30° C. for 3 days to grow colonies.Yeast total DNA are extracted by Zymoprep Yeast Plasmid Miniprep Kit(ZYMO research).

DNA Sequencing

E. coli transformation for DNA sequencing was carried out byelectroporation (BIO-RAD Gene Pulser). DNA Plasmids were prepared byalkaline method (Molecular Cloning, Cold Spring Harbor) or with theQiagen® Plasmid Kit. DNA fragments were recovered from agarose gel bythe Qiagen gel extraction Kit. PCR was performed using a PTC-200 DNAEngine. The ABI PRISM™ 310 Genetic Analyzer was used for determinationof all DNA sequences.

Construction of Protease Expression Vector

The Thermoascus M35 protease gene was amplified with the primer pairProt F (SEQ ID NO: 5) and Prot R (SEQ ID NO: 6). The resulting PCRfragments were introduced into S. cerevisiae YNG318 together with thepJC039 vector (described in WO 2001/92502) digested with restrictionenzymes to remove the Humicola insolens cutinase gene.

The Plasmid in yeast clones on SC-glucose plates was recovered toconfirm the internal sequence and termed as pJTP001.

Construction of Yeast Library and Site-Directed Variants

Library in yeast and site-directed variants were constructed by SOE PCRmethod (Splicing by Overlap Extension, see “PCR: A practical approach”,p. 207-209, Oxford University press, eds. McPherson, Quirke, Taylor),followed by yeast in vivo recombination.

General Primers for Amplification and Sequencing

The primers AM34 (SEQ ID NO: 7) and AM35 (SEQ ID NO:8) were used to makeDNA fragments containing any mutated fragments by the SOE methodtogether with degenerated primers (AM34+Reverse primer and AM35+forwardprimer) or just to amplify a whole protease gene (AM34+AM35).

PCR reaction system: Conditions: 48.5 microL H₂O 1 94° C. 2 min 2 beadspuRe Taq Ready-To-Go PCR 2 94° C. 30 sec (Amersham Biosciences) 0.5microL × 2 100 pmole/microL of primers 3 55° C. 30 sec 0.5 microLtemplate DNA 4 72° C. 90 sec 2-4 25 cycles 5 72° C. 10 min

DNA fragments were recovered from agarose gel by the Qiagen gelextraction Kit. The resulting purified fragments were mixed with thevector digest. The mixed solution was introduced into Saccharomycescerevisiae to construct libraries or site-directed variants by in vivorecombination.

Relative Activity Assay

Yeast clones on SC-glucose were inoculated to a well of a 96-well microtitre plate containing YPD+Zn medium and cultivated at 28° C. for 3days. The culture supernatants were applied to a 96-well zein microtiter plate and incubated at at least 2 temperatures (ex., 70° C. and80° C.) for more than 4 hours or overnight. The turbidity of zein in theplate was measured as A630 and the relative activity (higher/lowertemperatures) was determined as an indicator of thermoactivityimprovement. The clones with higher relative activity than the parentalvariant were selected and the sequence was determined.

Remaining Activity Assay

Yeast clones on SC-glucose were inoculated to a well of a 96-well microtitre plate and cultivated at 28° C. for 3 days. Protease activity wasmeasured at 65° C. using azo-casein (Megazyme) after incubating theculture supernatant in 20 mM sodium acetate buffer, pH 4.5, for 10 minat a certain temperature (80° C. or 84° C. with 4° C. as a reference) todetermine the remaining activity. The clones with higher remainingactivity than the parental variant were selected and the sequence wasdetermined.

Azo-Casein Assay

20 microL of samples were mixed with 150 microL of substrate solution (4mL of 12.5% azo-casein in ethanol in 96 mL of 20 mM sodium acetate, pH4.5, containing 0.01% triton-100 and 0.25 mM ZnSO₄) and incubated for 4hours or longer.

After adding 20 microL/well of 100% trichloroacetic acid (TCA) solution,the plate was centrifuge and 100 microL of supernatants were pipette outto measure A440.

Expression of Protease Variants in Aspergillus oryzae

The constructs comprising the protease variant genes were used toconstruct expression vectors for Aspergillus. The Aspergillus expressionvectors consist of an expression cassette based on the Aspergillus nigerneutral amylase II promoter fused to the Aspergillus nidulans triosephosphate isomerase non translated leader sequence (Pna2/tpi) and theAspergillus niger amyloglycosidase terminator (Tamg). Also present onthe plasmid was the Aspergillus selective marker amdS from Aspergillusnidulans enabling growth on acetamide as sole nitrogen source. Theexpression plasmids for protease variants were transformed intoAspergillus as described in Lassen et al., 2001, Appl. Environ.Microbiol. 67: 4701-4707. For each of the constructs 10-20 strains wereisolated, purified and cultivated in shake flasks.

Purification of Expressed Variants

-   -   1. Adjust pH of the 0.22 μm filtered fermentation sample to 4.0.    -   2. Put the sample on an ice bath with magnetic stirring. Add        (NH₄)₂SO₄ in small aliquots (corresponding to approx. 2.0-2.2 M        (NH₄)₂SO₄ not taking the volume increase into account when        adding the compound).    -   3. After the final addition of (NH₄)₂SO₄, incubate the sample on        the ice bath with gentle magnetic stirring for min. 45 min.    -   4. Centrifugation: Hitachi himac CR20G High-Speed Refrigerated        Centrifuge equipped with R20A2 rotor head, 5° C., 20,000 rpm, 30        min.    -   5. Dissolve the formed precipitate in 200 mL 50 mM Na-acetate pH        4.0.    -   6. Filter the sample by vacuum suction using a 0.22 micro m PES        PLUS membrane (IWAKI).    -   7. Desalt/buffer-exchange the sample to 50 mM Na-acetate pH 4.0        using ultrafiltration (Vivacell 250 from Vivascience equipped        with 5 kDa MWCO PES membrane) overnight in a cold room. Dilute        the retentate sample to 200 ml using 50 mM Na-acetate pH 4.0.        The conductivity of sample is preferably less than 5 mS/cm.    -   8. Load the sample onto a cation-exchange column equilibrated        with 50 mM Na-acetate pH 4.0. Wash unbound sample out of the        column using 3 column volumes of binding buffer (50 mM        Na-acetate pH 4.0), and elute the sample using a linear        gradient, 0-100% elution buffer (50 mM Na-acetate+1 M NaCl pH        4.0) in 10 column volumes.    -   9. The collected fractions are assayed by an endo-protease assay        (cf. below) followed by standard SDS-PAGE (reducing conditions)        on selected fractions. Fractions are pooled based on the        endo-protease assay and SDS-PAGE.

Endo-Protease Assay

-   -   1. Protazyme OL tablet/5 ml 250 mM Na-acetate pH 5.0 is        dissolved by magnetic stirring (substrate: endo-protease        Protazyme AK tablet from Megazyme—cat. #PRAK 11/08).    -   2. With stirring, 250 microL of substrate solution is        transferred to a 1.5 mL Eppendorf tube.    -   3. 25 microL of sample is added to each tube (blank is sample        buffer).    -   4. The tubes are incubated on a Thermomixer with shaking (1000        rpm) at 50° C. for 15 minutes.    -   5. 250 microL of 1 M NaOH is added to each tube, followed by        vortexing.    -   6. Centrifugation for 3 min. at 16,100×G and 25° C.    -   7. 200 microL of the supernatant is transferred to a MTP, and        the absorbance at 590 nm is recorded.

TABLE 1 Relative Activity of protease variants. Numbering ofsubstitution(s) starts from N-terminal of the mature peptide in aminoacids 1 to 177 of SEQ ID NO: 3. Remaining Activity VariantSubstitution(s) and/or deletion(s) 80° C. 84° C. JTP082AS5/D79L/S87P/A112P/D142L 53% JTP091 D79L/S87P/A112P/T124V/D142L 43%JTP092 AS5/N26R/D79L/S87P/A112P/D142L 60% JTP095N26R/T46R/D79L/S87P/A112P/D142L 62% JTP096 T46R/D79L/S87P/T116V/D142L67% JTP099 D79L/P81R/S87P/A112P/D142L 80% JTP101A27K/D79L/S87P/A112P/T124V/D142L 81% JTP116D79L/Y82F/S87P/A112P/T124V/D142L 59% JTP117D79L/Y82F/S87P/A112P/T124V/D142L 94% JTP127D79L/S87P/A112P/T124V/A126V/D142L 53%

TABLE 2 Relative Activity of protease variants. Numbering ofsubstitution(s) starts from N-terminal of the mature peptide in aminoacids 1 to 177 of SEQ ID NO: 3. Relative Activity 80° C./ 85° C./Variant Substitutions 70° C. 70° C. JTP050 D79L S87P A112P D142L 23% 9%JTP134 D79L Y82F S87P A112P D142L 40% JTP135 S38T D79L S87P A112P A126VD142L 62% JTP136 D79L Y82F S87P A112P A126V D142L 59% JTP137 A27K D79LS87P A112P A126V D142L 54% JTP145 S49P D79L S87P A112P D142L 59% JTP146S50P D79L S87P A112P D142L 63% JTP148 D79L S87P D104P A112P D142L 64%JTP161 D79L Y82F S87G A112P D142L 30% 12% JTP180 S70V D79L Y82F S87GY97W A112P 52% D142L JTP181 D79L Y82F S87G Y97W D104P A112P 45% D142LJTP187 S70V D79L Y82F S87G A112P D142L 45% JTP188 D79L Y82F S87G D104PA112P D142L 43% JTP189 D79L Y82F S87G A112P A126V D142L 46% JTP193 Y82FS87G S70V D79L D104P A112P 15% D142L JTP194 Y82F S87G D79L D104P A112PA126V 22% D142L JTP196 A27K D79L Y82F S87G D104P A112P 18% A126V D142L

TABLE 3 Relative Activity of protease variants. Numbering ofsubstitution(s) starts from N-terminal of the mature peptide in aminoacids 1 to 177 of SEQ ID NO: 3. Relative Activity 80° C./ VariantSubstitutions 70° C. JTP196 A27K D79L Y82F S87G D104P A112P 55% A126VD142L JTP210 A27K Y82F S87G D104P A112P A126V D142L 36% JTP211 A27K D79LY82F D104P A112P A126V D142L 44% JTP213 A27K Y82F D104P A112P A126VD142L 37%

Example 2 Temperature Profile of Selected Protease Variants UsingPurified Enzymes

Selected protease variants showing good thermostability were purifiedand the purified enzymes were used in a zein-BCA assay as describedbelow. The remaining protease activity was determined at 60° C. afterincubation of the enzyme at elevated temperatures as indicated for 60min.

Zein-BCA Assay:

Zein-BCA assay was performed to detect soluble protein quantificationreleased from zein by variant proteases at various temperatures.

Protocol:

-   -   1) Mix 10 microL of 10 micro g/mL enzyme solutions and 100        microL of 0.025% zein solution in a micro titer plate (MTP).    -   2) Incubate at various temperatures for 60 min.    -   3) Add 10 microL of 100% trichloroacetic acid (TCA) solution.    -   4) Centrifuge MTP at 3500 rpm for 5 m.    -   5) Take out 15 microL to a new MTP containing 100 microL of BOA        assay solution (Pierce Cat #:23225, BOA Protein Assay Kit).    -   6) Incubate for 30 min. at 60° C.    -   7) Measure A562.

The results are shown in Table 4. All of the tested protease variantsshowed an improved thermostability as compared to the wild type (WT)protease.

TABLE 4 Zein-BCA assay Sample incubated 60 min at indicated temperatures(° C.) (micro g/mL Bovine serum albumin equivalent peptide released)WT/Variant 60° C. 70° C. 75° C. 80° C. 85° C. 90° C. 95° C. WT(wild-type) 94 103 107 93 58 38 JTP050 86 101 107 107 104 63 36 (D79L +S87P + A112P + D142L) JTP077 82 94 104 105 99 56 31 (A27K + D79L +S87P + A112P + D142L) JTP188 71 83 86 93 100 75 53 (D79L + Y82F + S87G +D104P + A112P + D142L) JTP196 87 99 103 106 117 90 38 (A27K + D79L +Y82F + S87G + D104P + A112P + A126V + D142L)

Determination of Relative Activity for Proteases Using Azo Casein Assay

20 microL of samples containing approx. 0.01 mg/ml were mixed with 150microL of substrate solution (4 mL of 12. 5% azo-casein in ethanol in 96mL of 20 mM sodium acetate, pH 4. 5, containing 0. 01% triton-100 and 0.25 mM ZnSO₄) and incubated for 5 hours at 70° C. and 80° C.After adding 20 microL/well of 100% trichloroacetic acid (TCA) solution,the plate was centrifuge and 80 microL of supernatants were pipette outto measure A440.

Relative Activity Sample name 80° C./70° C. Protease RH  34% Protease TF106% Portease TA  19% Protease PF 154%

Example 4

Oil Extraction after Protease Treatment of Whole StillageIndustrially produced whole stillage collected from a first generationdry-grind corn ethanol plant (12-13% DS) was heated to 85° C., pH 4, ina water bath (Fisher Scientific IsoTemp 220) for two 15 hours.Approximately 25 grams of whole stillage was then aliquoted intopre-weighed 50 ml conical tubes (VWR 89039-660) and incubated anadditional two hours in the water bath. Enzymes were dosed according tothe specifications in Table 5 and the volume of stock solution to add tofermentation was found using the equation:

${{{Enz}.{dose}}({ml})} = \frac{\begin{matrix}{{Final}{{enz}.{dose}}\left( {{mg}{EP}/g{DS}} \right) \times} \\{{Mash}{weight}(g) \times {Solid}{content}\left( {\%{DS}} \right)}\end{matrix}}{{{Conc}.{enzyme}}\left( {{mg}{EP}/{ml}} \right)}$

TABLE 5 Enzyme doses Enzyme Dose Units 1 Control  0.00 μg ep/g DS 2 PF20.00 μg ep/g DS 3 RH 20.00 μg ep/g DS 4 TA 20.00 μg ep/g DS 5 TA 19620.00 μg ep/g DS 6 TF 20.00 μg ep/g DSWater was dosed into each sample such that the total added volume ofenzyme and water was ˜80 μL/25 g sample. Tubes were capped and placed ina hybridization rotisserie oven (Boekel Big Shot III Model #230402) setat 85° C. with rotation (setting #14) for 2 hours. After incubation,tubes were cooled to room temperature then weighed prior to oilextraction. Hexane was added to each sample at a dose of 0.125 mLhexane/1 g of whole stillage material. Each tube was covered inDura-seal to prevent sample leakage, and mixed thoroughly. Tubes werecentrifuged at 3,000×g for 10 minutes in an Avanti JE Series centrifugewith JS-5.3 rotor (Beckmann Coulter). After centrifugation, theoil/hexane layer (supernatant) was removed using a positive displacementpipette (Ranin MR-250 and MR-1000), transferred to a pre-weighed 5 mLflip-top tube (Fisherbrand 03-338-1C), and reweighed. The density of thesample was measured using a Rudolph Research Analytical density meter(DDM 2911). The density of the supernatant was then inserted into thestandard curve equation derived by numerous percent oil in hexanemixtures measured on the densitometer to determine the percent oil inthe supernatant.

(ρ₀+γ−ρ_(h))/(2*γ)−sqrt((ρ_(o)+γ−ρ_(h)){circumflex over( )}2+4*γ*ρ_(h)−4*γ*φ/(2*γ)=% oil

-   -   ρo=density of oil    -   ρh=density of hexane    -   γ=excess molar volume coefficient        From this value the total percent oil in the starting material        (whole stillage) was derived. Results are displayed in FIG. 2        (Control=no enzyme; Protease PF; Protease RH; Protease TA;        Protease TA196; Protease TF).

Conclusions

Addition of proteases was found to have a positive significant impact oncorn oil extraction from whole stillage. The results show astatistically higher extraction of corn oil for Protease PF, Protease TA196, and Protease TF, 25%, 17%, and 16%, respectively, higher than theControl.

Example 5

Oil Extraction from Liquefied Corn Mash Using Protease

Four slurries were prepared by combining industrially produced groundcorn and- backset from a first generation dry-grind ethanol plant andtap water to a target total weight of 180 g at 32.50% Dry Solids (DS).Initial slurry pH was approximately 5.1 and was adjusted with either 45%KOH or 40% v/v H₂SO₄ to pH 5.8. Enzymes were dosed according to thespecifications in Table 6 and the volume of stock solution to add tofermentation was found using the equation:

${{{Enz}.{dose}}({ml})} = \frac{\begin{matrix}{{Final}{{enz}.{dose}}\left( {{mg}{EP}/g{DS}} \right) \times} \\{{Slurry}{weight}(g) \times {Solid}{content}\left( {\%{DS}} \right)}\end{matrix}}{{{Conc}.{enzyme}}\left( {{mg}{EP}/{ml}} \right)}$

TABLE 6 Experimental Plan Amylase Dose Units Protease Dose Units ControlLSCDS 0.02 % w/w corn 1 LSCDS 0.02 % w/w corn PF 2.5 μg EP/g DS 2 LSCDS0.02 % w/w corn PF 5.0 μg EP/g DSLiquefactions took place in a Labomat BFA-24 (Mathis, Concord, NC) usingthe following parameters: 6° C./min. Ramp, 15 minute Ramp to 80° C.,hold for 1 min, Ramp to 85° C. at 1° C./min and holding for 103 min., 40rpm for 30 seconds to the left and 30 seconds to the right. Followingliquefaction, all canisters were cooled in an ice bath to roomtemperature then transferred to a beaker and stirred. The dry solidscontent of each mash was measured on a HB43-S moisture balance(Mettler-Toledo, Cleveland, OH). Approximately 25 grams of liquefact wasthen aliquoted into pre-weighed 50 ml conical tubes (VWR 89039-660).Hexane was added to each sample at a dose of 0.125 mL hexane/1 g ofliquefact material. Each tube was covered in Duraseal to prevent sampleleakage, and mixed thoroughly. Tubes were centrifuged at 3,000×g for 10minutes in an Avanti JE Series centrifuge with JS-5.3 rotor (BeckmannCoulter). After centrifugation, the oil/hexane layer (supernatant) wasremoved using a positive displacement pipette (Ranin MR-250 andMR-1000), transferred to a pre-weighed 5 mL flip-top tube (Fisherbrand03-338-1C), and reweighed. The density of the sample was measured usinga Rudolph Research Analytical density meter (DDM 2911). The density ofthe supernatant was then inserted into a standard curve equation derivedby numerous percent oil in hexane mixtures measured on the densitometerto determine the percent oil in the supernatant.

(ρ_(o)+γ−ρ_(h))/(2*γ)−sqrt((ρ_(o)+γ−ρ_(h)){circumflex over( )}2+4*γ*ρ_(h)−4*γ*φ/(2*γ)=% oil

-   -   ρo=density of oil    -   ρh=density of hexane    -   γ=excess molar volume coefficient        From this value the total percent oil in the starting material        (liquefact) was derived. Results are displayed in FIG. 3        (Control=no enzyme; Protease PF).

Conclusions:

Addition of Protease PF was found to have a positive significant impacton corn oil extraction from liquefied material. The results show astatistically different higher extraction of corn oil for PF between 16and 24%.

Example 6

Oil Extraction after Protease Treatment of Evaporated Centrate (Syrup)Industrially produced syrup collected from a first generation dry-grindcorn ethanol plant (8-40% DS) was heated to 85° C., pH 4, in a waterbath (Fisher Scientific IsoTemp 220) for two hours. Approximately 25grams of syrup was then aliquoted into pre-weighed 50 ml conical tubes(VWR 89039-660) and incubated an additional two hours in the water bath.Enzymes were dosed according to the specifications in Table 7 and thevolume of stock solution to add to fermentation was found using theequation:

${{{Enz}.{dose}}({ml})} = \frac{\begin{matrix}{{Final}{{enz}.{dose}}\left( {{mg}{EP}/g{DS}} \right) \times} \\{{Mash}{weight}(g) \times {Solid}{content}\left( {\%{DS}} \right)}\end{matrix}}{{{Conc}.{enzyme}}\left( {{mg}{EP}/{ml}} \right)}$

TABLE 7 Enzyme doses Enzyme Dose Units 1 Control  0.00 μg ep/g DS 2 PF20.00 μg ep/g DS 3 RH 20.00 μg ep/g DS 4 TA 20.00 μg ep/g DS 5 TA 19620.00 μg ep/g DS 6 TF 20.00 μg ep/g DSWater was dosed into each sample such that the total added volume ofenzyme and water was ˜90 μL/25 g sample. Tubes were covered and placedin a water bath set at 85° C. for 2 hours with vortexing every 15minutes. After incubation, tubes were cooled to room temperature thenweighed prior to oil extraction. Hexane was added to each sample at adose of 0.18 mL hexane/1 g of syrup material. Each tube was covered inDura-seal to prevent sample leakage, and mixed thoroughly. Tubes werecentrifuged at 3,000×g for 10 minutes in an Avanti JE Series centrifugewith JS-5.3 rotor (Beckmann Coulter). After centrifugation, theoil/hexane layer (supernatant) was removed using a positive displacementpipette (Ranin MR-250 and MR-1000), transferred to a pre-weighed 5 mLflip-top tube (Fisherbrand 03-338-1C), and reweighed. The density of thesample was measured using a Rudolph Research Analytical density meter(DDM 2911). The density of the supernatant was then inserted into thestandard curve equation derived by numerous percent oil in hexanemixtures measured on the densitometer to determine the percent oil inthe supernatant.

(ρ_(o)+γ−ρ_(h))/(2*γ)−sqrt((ρ_(o)+γ−ρ_(h)){circumflex over( )}2+4*γ*ρ_(h)−4*γ*ρ)/(2*γ)=% oil

-   -   Where ρ is the measured density        From this value the total percent oil in the starting material        (syrup) was derived. Results are displayed in FIG. 4 (Control=no        enzyme; Protease PF; Protease RH; Protease TA; Protease TA196;        Protease TF).

Conclusions

Addition of proteases was found to have a positive significant impact oncorn oil extraction from syrup. The results show a statistically higherextraction of corn oil for Protease TF, and Protease PF, 2.9%, and 2.4%,respectively, higher than the Control.

Example 7

Oil Extraction from Liquefied Corn Mash Using Protease

Eighteen slurries were prepared by combining industrially producedground corn and backset from a first generation dry-grind ethanol plantand tap water to a target total weight of 30 g at 32.50% Dry Solids(DS). Initial slurry pH was approximately 5.1 and not adjusted. Enzymeswere dosed according to the specifications in Table 8 and the volume ofstock solution to add to fermentation was found using the equation:

${{{Enz}.{dose}}({ml})} = \frac{\begin{matrix}{{Final}{{enz}.{dose}}\left( {{mg}{EP}/g{DS}} \right) \times} \\{{Slurry}{weight}(g) \times {Solid}{content}\left( {\%{DS}} \right)}\end{matrix}}{{{Conc}.{enzyme}}\left( {{mg}{EP}/{ml}} \right)}$

Water was dosed into each sample such that the total added volume ofenzyme and water was approx.. 230 μL/30 g sample. Tubes were coveredwith a strip of Dura-seal, capped and placed in a water bath set at 85°C. for 2 hours with vortexing every 15 minutes. After incubation, tubeswere cooled in a room temperature water bath for 15 minutes then weighedprior to oil extraction. Hexane was added to each sample at a dose of0.125 mL hexane/1 g of liquefied material. Each tube was covered inDura-seal to prevent sample leakage, and mixed thoroughly. Tubes werecentrifuged at 3,000×g for 10 minutes in an Avanti JE Series centrifugewith JS-5.3 rotor (Beckmann Coulter). After centrifugation, theoil/hexane layer (supernatant) was removed using a positive displacementpipette (Ranin MR-250 and MR-1000), transferred to a pre-weighed 5 mLflip-top tube (Fisherbrand 03-338-AC), and reweighed. The density of thesample was measured using a Rudolph Research Analytical density meter(DDM 2911). The density of the supernatant was then inserted into astandard curve equation derived by numerous percent oil in hexanemixtures measured on the densitometer to determine the percent oil inthe supernatant.

(ρ_(o)+γ−ρ_(h))/(2*γ)−sqrt((ρ_(o)+γ−ρ_(h)){circumflex over( )}2+4*γ*ρ_(h)−4*γ*ρ)/(2*γ)=% oil

-   -   ρo=density of oil    -   ρh=density of hexane    -   γ=excess molar volume coefficient        From this value the total % oil in the starting material        (liquefact) was derived. Results are displayed in FIG. 5        (Control=no enzyme; Protease PF; Protease RH; Protease TA;        Protease TA196; Protease TF).

TABLE 8 Experimental Plan Alpha- Amylase Dose Units Protease Dose UnitsControl 369 0.02 % w/w corn  0 μg EP/g DS Control 369 0.02 % w/w corn  0μg EP/g DS Control 369 0.02 % w/w corn  0 μg EP/g DS  1 369 0.02 % w/wcorn PF 20 μg EP/g DS  2 369 0.02 % w/w corn PF 20 μg EP/g DS  3 3690.02 % w/w corn PF 20 μg EP/g DS  4 369 0.02 % w/w corn TA 20 μg EP/g DS 5 369 0.02 % w/w corn TA 20 μg EP/g DS  6 369 0.02 % w/w corn TA 20 μgEP/g DS  7 369 0.02 % w/w corn TA196 20 μg EP/g DS  8 369 0.02 % w/wcorn TA196 20 μg EP/g DS  9 369 0.02 % w/w corn TA196 20 μg EP/g DS 10369 0.02 % w/w corn TF 20 μg EP/g DS 11 369 0.02 % w/w corn TF 20 μgEP/g DS 12 369 0.02 % w/w corn TF 20 μg EP/g DS 13 369 0.02 % w/w cornRH 20 μg EP/g DS 14 369 0.02 % w/w corn RH 20 μg EP/g DS 15 369 0.02 %w/w corn RH 20 μg EP/g DS

Conclusions

Addition of protease was found to have a positive significant impact oncorn oil extraction from liquefied mash. The results show astatistically higher extraction of corn oil for Protease PF, ProteaseTA196, Protease TF and Protease TA, 171.1%, 143.4%, 107.3%, and 80.9%,respectively, higher than the Control.

The present invention is further described in the following numberedparagraphs:1. A process of recovering oil, comprising

-   -   (a) converting a starch-containing material into dextrins with        an alpha-amylase; optionally recovering oil during and/or after        step (a)    -   (b) saccharifying the dextrins using a carbohydrate source        generating enzyme to form a sugar;    -   (c) fermenting the sugar in a fermentation medium into a        fermentation product using a fermenting organism;    -   (d) recovering the fermentation product to form a whole        stillage;    -   (e) separating the whole stillage into thin stillage and wet        cake;    -   (e′) optionally concentrating the thin stillage into syrup;    -   (f) recovering oil from the thin stillage and/or optionally the        syrup, wherein a protease having a thermostability value of more        than 20% determined as Relative Activity at 80° C./70° C. is        present and/or added during step (a) or steps (d)-(e′).        2. The process of paragraph 1, wherein the protease is present        in and/or added in starch-containing material converting step        (a).        3. The process of paragraphs 1-2, wherein saccharification        step b) and fermentation step c) are carried out simultaneously        or sequentially.        4. The process of any of paragraphs 1-3, wherein        starch-containing material is converted to dextrins by        liquefaction.        5. The process of any of paragraphs 1-4, wherein the temperature        in step (a) is above the initial gelatinization temperature,        such as at a temperature between 80-90° C., such as around 85°        C.        6. The process of paragraphs 4-5, wherein a jet-cooking step is        carried out before in step (a).        7. The process of paragraph 6, wherein jet-cooking is carried        out at a temperature between 95-140° C. for about 1-15 minutes,        preferably for about 3-10 minutes, especially around about 5        minutes.        8. The process of any of paragraphs 1-7, wherein the pH in        step (a) is from 4-7, preferably 4.5-6.        9. The process of any of paragraphs 1-8, further comprising,        before step (a), the steps of:    -   i) reducing the particle size of the starch-containing material,        preferably by dry milling;    -   ii) forming a slurry comprising the starch-containing material        and water.        10. The process of any of paragraphs 1-9, further comprising a        pre-saccharification step, before saccharification step (b),        carried out for 40-90 minutes at a temperature between 30-65° C.        11. The process of any of paragraphs 1-10, wherein        saccharification is carried out at a temperature from 20-75° C.,        preferably from 40-70° C., such as around 60° C., and at a pH        between 4 and 5.        12. The process of any of paragraphs 1-11, wherein fermentation        step (c) or simultaneous saccharification and fermentation (SSF)        (i.e., steps (b) and (c)) are carried out at a temperature from        25° C. to 40° C., such as from 28° C. to 35° C., such as from        30° C. to 34° C., preferably around about 32° C.        13. The process of any of paragraphs 1-12, wherein fermentation        step (c) or simultaneous saccharification and fermentation (SSF)        (i.e., steps (b) and (c)) are ongoing for 6 to 120 hours, in        particular 24 to 96 hours.        14. The process of any of paragraphs 1-13, wherein        starch-containing material converting step (a), saccharification        step (b) and fermentation step (c) are carried out        simultaneously or sequentially.        15. The process of paragraph 14, wherein starch-containing        material converting step (a) is carried out at a temperature        below the initial gelatinization temperature, preferably from        20-60° C., such as 25-40° C., such as around 30-35° C., such as        around 32° C.        16. The process of paragraph 15, wherein the starch-containing        material is converted to dextrins and the dextrins are        saccharified to a sugar by treating the starch-containing        material with an alpha-amylase and glucoamylase below the        initial gelatinization temperature of the starch-containing        material.        17. The process of any of paragraphs 15 or 16, wherein the        conversion of the starch-containing material to dextrins, the        saccharification of the dextrins to sugars, and the fermentation        of the sugars are carried out in a single step.        18. The process of any of paragraphs 14-17, wherein the        glucoamylase is derived from a strain of Trametes, such as a        strain of Trametes cingulata, or a strain of Althelia, such as a        strain of Athelia rolfsii.        19. The process of any of paragraphs 14-18, wherein the        alpha-amylase is derived from a strain Rhizomucor, such as a        strain of Rhizomucor pusillus, such as a Rhizomucor pusillus        alpha-amylase with a starch-binding domain (SBD), such as a        Rhizomucor pusillus alpha-amylase with Aspergillus niger        glucoamylase linker and SBD.        20. The process of any of paragraphs 14-19, wherein the        starch-containing material is granular starch.        21. The process of any of paragraphs 14-20, wherein the        starch-containing material is reducing the particle size,        preferably by milling, to 0.05 to 3.0 mm, preferably 0.1-0.5 mm.        22. The process of any of paragraphs 14-21, wherein simultaneous        saccharification and fermentation (SSF) is carried out so that        the sugar level, such as glucose level, is kept below 6 wt.-%,        preferably below about 3 wt.-%, preferably below about 2 wt.-%,        more preferred below about 1 wt.-%., even more preferred below        about 0.5%, or even more preferred 0.25% wt.-%, such as below        about 0.1 wt.-%.        23. The process of any of paragraphs 14-22, wherein the pH in is        from 4-7, preferably 4.5-6 when conversion of the        starch-containing material to dextrins, the saccharification of        the dextrins to a sugar, and the fermentation of the sugar are        carried out in a single step.        24. The process of any of paragraphs 1-23, wherein        starch-containing material in step (a), including granular        starch, contains 20-55 wt.-% dry solids, preferably 25-40 wt.-%        dry solids, more preferably 30-35% dry solids.        25. The process of any of paragraphs 1-24, wherein the protease        is present in and/or added to the whole stillage in step (d)        and/or the thin stillage in or after separation in step (e),        and/or syrup in step (e′).        26. The process of any of paragraphs 1-25, wherein separation in        step (e) is carried out by centrifugation, preferably a decanter        centrifuge, filtration, preferably using a filter press, a screw        press, a plate-and-frame press, a gravity thickener or decker.        27. The process of any of paragraphs 1-26, wherein the        starch-containing material is cereal.        28. The process of any of paragraphs 1-27, wherein the        starch-containing material is selected from the group consisting        of corn, wheat, barley, cassava, sorghum, rye, potato, beans,        milo, peas, rice, sago, sweet potatoes, tapioca, or any        combination thereof.        29. The process of any of paragraphs 1-28, wherein the        fermentation product is selected from the group consisting of        alcohols (e.g., ethanol, methanol, butanol, 1,3-propanediol);        organic acids (e.g., citric acid, acetic acid, itaconic acid,        lactic acid, gluconic acid, gluconate, lactic acid, succinic        acid, 2,5-diketo-D-gluconic acid); ketones (e.g., acetone);        amino acids (e.g., glutamic acid); gases (e.g., H2 and CO2), and        more complex compounds, including, for example, antibiotics        (e.g., penicillin and tetracycline); enzymes; vitamins (e.g.,        riboflavin, B12, beta-carotene); and hormones.        30. The process of any of paragraphs 1-29, wherein the        fermentation product is ethanol.        31. The process of any of paragraphs 1-30, wherein the        carbohydrate source generating enzyme in step (b) is a        glucoamylase.        32. The process of any of paragraphs 1-31, wherein the        glucoamylase present and/or added during saccharification and/or        fermentation is of fungal origin, preferably from a strain of        Aspergillus, preferably A. niger, A. awamori, or A. oryzae; or a        strain of Trichoderma, preferably Trichoderma reesei; or a        strain of Talaromyces, preferably Talaromyces emersonii, or a        strain of Pycnoporus, or a strain of Gloeophyllum.        33. The process of any of paragraphs 1-32, wherein the        fermentation product is recovered by distillation.        34. The process of recovering oil of any of paragraphs 1-33,        comprising    -   (a) converting a starch-containing material into dextrins with        an alpha-amylase at a temperature above the initial        gelatinization temperature;    -   (b) saccharifying the dextrins using a carbohydrate source        generating enzyme to form a sugar;    -   (c) fermenting the sugar in a fermentation medium into a        fermentation product using a fermenting organism;    -   (d) recovering the fermentation product to form a whole        stillage;    -   (e) separating the whole stillage into thin stillage and wet        cake;    -   (e′) optionally concentrating the thin stillage into syrup;    -   (f) recovering oil from the thin stillage and/or optionally the        syrup, wherein a protease having a thermostability value of more        than 20% determined as Relative Activity at 80° C./70° C. is        present and/or added during step (a) or any of paragraphs        (d)-(e′).        35. The process of paragraph 34, wherein the temperature during        step (a) is between 80-90° C., such as around 85° C.        36. The process of recovering oil of any of paragraphs 1-35,        comprising    -   (a) converting a starch-containing material into dextrins with        an alpha-amylase at a temperature below the initial        gelatinization temperature;    -   (b) saccharifying the dextrins using a carbohydrate source        generating enzyme to form a sugar;    -   (c) fermenting the sugar in a fermentation medium into a        fermentation product using a fermenting organism;    -   (d) recovering the fermentation product to form a whole        stillage;    -   (e) separating the whole stillage into thin stillage and wet        cake;    -   (e′) optionally concentrating the thin stillage into syrup;    -   (f) recovering oil from the thin stillage and/or optionally the        syrup, wherein a protease having a thermostability value of more        than 20% determined as Relative Activity at 80° C./70° C. is        present and/or added during any of step (d)-(e′).        37. The process of paragraph 36, wherein the temperature during        step (a) is from 25° C. to 40° C., such as from 28° C. to 35°        C., such as from 30° C. to 34° C., preferably around about 32°        C.        38. The process of any of paragraphs 1-37, wherein the oil is        recovered from the thin stillage and/or syrup/evaporated        centrate, e.g., by extraction, such as hexane extraction.        39. The process of any of paragraphs 1-38, wherein the protease        has a thermostability of more than 30%, more than 40%, more than        50%, more than 60%, more than 70%, more than 80%, more than 90%        more than 100%, such as more that 10⁵%, such as more than 110%,        such as more than 115%, such as more than 120% determined as        Relative Activity at 80° C./70° C.        40. The process of any of paragraphs 1-39, wherein the protease        has a thermostability between 50 and 115%, such as between 50        and 70%, such as between 50 and 60%, such as between 100 and        120%, such as between 10⁵ and 115% determined as Relative        Activity at 80° C./70° C.        41. The process of any of paragraphs 1-40, wherein the protease        has a thermostability of more than 12%, more than 14%, more than        16%, more than 18%, more than 20%, more than 25%, more than 30%,        more than 40%, more that 50%, more than 60%, more than 70%, more        than 80%, more than 90%, more than 100%, more than 110%        determined as Relative Activity at 85° C./70° C.        42. The process of any of paragraphs 1-41, wherein the protease        has a thermostability between 10 and 50%, such as between 10 and        30%, such as between 10 and 25% determined as Relative Activity        at 85° C./70° C.        43. The process of any of paragraphs 1-42, wherein the protease        has a thermostability between 50 and 110%, such as between 70        and 110%, such as between 90 and 110% determined as Relative        Activity at 85° C./70° C.        44. The process of any of paragraphs 1-43, wherein the protease        is of fungal origin.        45. The process of any of paragraphs 1-44, wherein the protease        is a variant of the metallo protease derived from a strain of        the genus Thermoascus, preferably a strain of Thermoascus        aurantiacus, especially Thermoascus aurantiacus CGMCC No. 0670.        46. The process of any of paragraphs 1-45, wherein the protease        is a variant of the metallo protease disclosed as the mature        part of SEQ ID NO: 2 disclosed in WO 2003/048353 or SEQ ID NO: 3        herein.        47. The process of any of paragraphs 1-46, wherein the parent        protease has at least 70%, such as at least 75% identity        preferably at least 80%, more preferably at least 85%, more        preferably at least 90%, more preferably at least 91%, more        preferably at least 92%, even more preferably at least 93%, most        preferably at least 94%, and even most preferably at least 95%,        such as even at least 96%, at least 97%, at least 98%, at least        99%, such as least 100% identity to the mature part of the        polypeptide of SEQ ID NO: 2 disclosed in WO 2003/048353 or SEQ        ID NO: 3 herein.        48. The process of any of paragraphs 1-47, wherein the protease        variant has at least 70%, such as at least 75% identity        preferably at least 80%, more preferably at least 85%, more        preferably at least 90%, more preferably at least 91%, more        preferably at least 92%, even more preferably at least 93%, most        preferably at least 94%, and even most preferably at least 95%,        such as even at least 96%, at least 97%, at least 98%, at least        99%, but less than 100% identity to the mature part of the        polypeptide of SEQ ID NO: 2 disclosed in WO 2003/048353 or SEQ        ID NO: 3 herein.        49. The process of any of paragraphs 1-48, wherein the protease        is a variant of the Thermoascus aurantiacus protease shown in        SEQ ID NO: 3 herein with the mutations selected from the group        consisting of:    -   A27K+D79L+Y82F+S87G+D104P+A112P+A126V+D142L;    -   D79L+Y82F+S87G+A112P+D142L;    -   Y82F+S87G+S70V+D79L+D104P+A112P+D142L;    -   Y82F+S87G+D79L+D104P+A112P+A126V+D142L.        50. The process of any of paragraphs 1-49, wherein the protease        is derived from Rhizomucor, such as Rhizomucor miehei, such as        the protease shown in SEQ ID NO: 9 or one having a sequence        identity thereto of at least 60%, at least 70%, at least 80%, at        least 90%, at least 95%, at least 96%, at least 97%, at least        98%, at least 99%.        51. The process of any of paragraphs 1-50, wherein the protease        is added in a concentration of 0.01-100, such 0.1-10 micro g/g        DS.        52. The process of any of paragraphs 1-51, wherein the protease        is of bacterial origin.        53. The process of any of paragraphs 1-52, wherein the protease        is derived from a strain of Pyrococcus, preferably a strain of        Pyrococcus furiosus.        54. The process of any of paragraphs 1-53, wherein the protease        is the one shown in SEQ ID NO: 1 in U.S. Pat. No. 6,258,726 or        SEQ ID NO: 4 herein.        55. The process of any of paragraphs 1-54, wherein the protease        has at least 60%, such as at least 70%, such as at least 80%,        such as at least 85%, such as at least 90%, such as at least        95%, such as at least 96%, such as at least 97%, such as at        least 98%, such as at least 99% identity to SEQ ID NO: 1 in U.S.        Pat. No. 6,258,726 or SEQ ID NO: 4 herein.        56. The process of any of paragraphs 1-54, wherein the protease        is a bacterial serine protease, such as derived from a strain of        Thermobifida, such as Thermobifida fusca, such as the protease        shown in SEQ ID NO: 10 or one having a sequence identity thereto        of at least 60%, at least 70%, at least 80%, at least 90%, at        least 95%, at least 96%, at least 97%, at least 98%, at least        99%.        57. The process of any of paragraphs 1-56, wherein the        alpha-amylase in step (a) is a bacterial alpha-amylase.        58. The process of any of paragraphs 1-57, wherein the bacterial        alpha-amylase is derived from the genus Bacillus, such as a        strain of Bacillus stearothermophilus, in particular a variant        of a Bacillus stearothermophilus alpha-amylase, such as the one        shown in SEQ ID NO: 3 in WO 99/019467 or SEQ ID NO: 1 herein, in        particular the Bacillus stearothermophilus alpha-amylase is        truncated, preferably to have from 485-495 amino acids, such as        around 491 amino acids.        59. The process of any of paragraphs 1-58, wherein the        alpha-amylase is selected from the group of Bacillus        stearothermophilus alpha-amylase variants comprising a double        deletion, such as I181*+G182*, or I181*+G182*+N193F (using SEQ        ID NO: 1 for numbering).        60. The process of any of paragraphs 1-59, wherein the        alpha-amylase is selected from the group of Bacillus        stearothermophilus alpha-amylase variants:    -   I181*+G182*+N193F+E129V+K177L+R179E;    -   181*+G182*+N193F+V59A+Q89R+E129V+K177L+R179E+H208Y+K220P+N224L+Q254S;    -   I181*+G182*+N193F+V59A Q89R+E129V+K177L+R179E+Q254S+M284V; and    -   I181*+G182*+N193F+E129V+K177L+R179E+K220P+N224L+S242Q+Q254S        (using SEQ ID NO: 1 for numbering).        61. The process of any of paragraphs 1-60 wherein oil is        extracted/recovered from the liquefied starch-containing        material during and/or after step (a), before saccharification        in step (b).        62. The process of recovering oil of any of paragraphs 1-61,        comprising    -   (c) converting a starch-containing material into dextrins with        an alpha-amylase;        -   recovering oil during and/or after step (a)    -   (b) saccharifying the dextrins using a carbohydrate source        generating enzyme to form a sugar;    -   (c) fermenting the sugar in a fermentation medium into a        fermentation product using a fermenting organism; wherein a        protease having a thermostability value of more than 20%        determined as Relative Activity at 80° C./70° C. is present        and/or added in step (a).        62. The process of recovering oil of any of paragraphs 1-61,        comprising    -   (a) converting a starch-containing material into dextrins with        an alpha-amylase;    -   (b) saccharifying the dextrins using a carbohydrate source        generating enzyme to form a sugar;    -   (c) fermenting the sugar in a fermentation medium into a        fermentation product using a fermenting organism;    -   (d) recovering the fermentation product to form a whole        stillage;    -   (e) separating the whole stillage into thin stillage and wet        cake;    -   (e′) concentrating the thin stillage into syrup;    -   (f) recovering oil from the syrup, wherein a protease having a        thermostability value of more than 20% determined as Relative        Activity at 80° C./70° C. is present and/or added in step (e′).        63. The process of paragraph 62, wherein the protease is derived        from a strain of Pyrococcus, preferably a strain of Pyrococcus        furiosus.        64. The process of any of paragraphs 62 or 63, wherein the        protease is the one shown in SEQ ID NO: 1 in U.S. Pat. No.        6,258,726 or SEQ ID NO: 4 herein.        65. The process of any of paragraphs 62-64, wherein the protease        has at least 60%, such as at least 70%, such as at least 80%,        such as at least 85%, such as at least 90%, such as at least        95%, such as at least 96%, such as at least 97%, such as at        least 98%, such as at least 99% identity to SEQ ID NO: 1 in U.S.        Pat. No. 6,258,726 or SEQ ID NO: 4 herein.        66. The process of paragraph 62, wherein the protease is a        bacterial serine protease, such as derived from a strain of        Thermobifida, such as Thermobifida fusca, such as the protease        shown in SEQ ID NO: 10 or one having a sequence identity thereto        of at least 60%, at least 70%, at least 80%, at least 90%, at        least 95%, at least 96%, at least 97%, at least 98%, at least        99%.        67. The process of paragraph 62, wherein the protease is of        fungal origin.        68. The process of any of paragraphs 62-67, wherein the protease        is a variant of the metallo protease derived from a strain of        the genus Thermoascus, preferably a strain of Thermoascus        aurantiacus, especially Thermoascus aurantiacus CGMCC No. 0670.        69. The process of any of paragraphs 62-68, wherein the protease        is a variant of the metallo protease disclosed as the mature        part of SEQ ID NO: 2 disclosed in WO 2003/048353 or SEQ ID NO: 3        herein.        70. The process of any of paragraphs 62-69, wherein the parent        protease has at least 70%, such as at least 75% identity        preferably at least 80%, more preferably at least 85%, more        preferably at least 90%, more preferably at least 91%, more        preferably at least 92%, even more preferably at least 93%, most        preferably at least 94%, and even most preferably at least 95%,        such as even at least 96%, at least 97%, at least 98%, at least        99%, such as least 100% identity to the mature part of the        polypeptide of SEQ ID NO: 2 disclosed in WO 2003/048353 or SEQ        ID NO: 3 herein.        71. The process of any of paragraphs 62-70, wherein the protease        variant has at least 70%, such as at least 75% identity        preferably at least 80%, more preferably at least 85%, more        preferably at least 90%, more preferably at least 91%, more        preferably at least 92%, even more preferably at least 93%, most        preferably at least 94%, and even most preferably at least 95%,        such as even at least 96%, at least 97%, at least 98%, at least        99%, but less than 100% identity to the mature part of the        polypeptide of SEQ ID NO: 2 disclosed in WO 2003/048353 or SEQ        ID NO: 3 herein.        72. The process of any of paragraphs 62-71, wherein the protease        is a variant of the Thermoascus aurantiacus protease shown in        SEQ ID NO: 3 herein with the mutations selected from the group        consisting of:    -   A27K+D79L+Y82F+S87G+D104P+A112P+A126V+D142L;    -   D79L+Y82F+S87G+A112P+D142L;    -   Y82F+S87G+S70V+D79L+D104P+A112P+D142L;    -   Y82F+S87G+D79L+D104P+A112P+A126V+D142L.        73. The process of any of paragraphs 62-72, wherein the protease        is derived from Rhizomucor, such as Rhizomucor miehei, such as        the protease shown in SEQ ID NO: 9 or one having a sequence        identity thereto of at least 60%, at least 70%, at least 80%, at        least 90%, at least 95%, at least 96%, at least 97%, at least        98%, at least 99%.        74. The process of any of paragraphs 62-73, wherein the protease        is added in a concentration of 0.01-100, such 0.1-10 micro g/g        DS.        75. A process of recovering oil, comprising    -   (a) converting a starch-containing material into dextrins with        an alpha-amylase;    -   (b) saccharifying the dextrins using a carbohydrate source        generating enzyme to form a sugar;    -   (c) fermenting the sugar in a fermentation medium into a        fermentation product using a fermenting organism;    -   (d) optionally recovering the fermentation product to form a        whole stillage;    -   (e) optimally separating the whole stillage into thin stillage        and wet cake;    -   (e′) optionally concentrating the thin stillage into syrup;        wherein a protease having a thermostability value of more than        20% determined as Relative Activity at 80° C./70° C. is present        and/or added in step (a) and/or steps (d)-(e′) and oil is        recovered during and/or after step (a).        76. A process of recovering oil, comprising    -   (a) converting a starch-containing material into dextrins with        an alpha-amylase;    -   (b) saccharifying the dextrins using a carbohydrate source        generating enzyme to form a sugar;    -   (c) fermenting the sugar in a fermentation medium into a        fermentation product using a fermenting organism;    -   (d) recovering the fermentation product;        wherein a protease having a thermostability value of more than        20% determined as Relative Activity at 80° C./70° C. is present        and/or added in step (a) and oil is recovered during and/or        after step (a).        77. A process of recovering oil, comprising    -   (a) converting a starch-containing material into dextrins with        an alpha-amylase and Pyrococcus furiosus protease;    -   (b) saccharifying the dextrins using a carbohydrate source        generating enzyme to form a sugar;    -   (c) fermenting the sugar in a fermentation medium into a        fermentation product using a fermenting organism;    -   (d) recovering the fermentation product, wherein oil is        recovered during and/or after step (a).        78. Use of a protease having a thermostability value of more        than 20%, such as more than 30%, more than 40%, more than 50%,        more than 60%, more than 70%, more than 80%, more than 90% more        than 100%, such as more that 10⁵%, such as more than 110%, such        as more than 115%, such as more than 120% determined as Relative        Activity at 80° C./70° C. for increasing oil recovery yields        from thin stillage and/or syrup/evaporated centrate in a        fermentation product production process.

1-20. (canceled)
 21. A process of recovering oil, comprising: (a)liquefying a starch-containing material into dextrins with analpha-amylase at a temperature above the initial gelatinizationtemperature, wherein the starch-containing material comprises corn; (b)saccharifying the dextrins using a glucoamylase to form a sugar; (c)fermenting the sugar in a fermentation medium into a fermentationproduct using a fermenting organism; (d) recovering the fermentationproduct to form a whole stillage; (e) separating the whole stillage intothin stillage and wet cake; (e′) optionally concentrating the thinstillage into syrup; and (f) recovering oil from the thin stillageand/or optionally the syrup, wherein a protease derived fromThermobifida is present or added during step (a).
 22. The process ofclaim 21, wherein the oil is recovered during and/or after step (a). 23.The process of claim 22, wherein oil recovery is increased higher thancontrol.
 24. The process of claim 21, wherein the oil is recovered fromthe thin stillage.
 25. The process of claim 24, wherein oil recovery isincreased higher than control.
 26. The process of claim 1, wherein theoil is optionally recovered from the syrup.
 27. The process of claim 26,wherein oil recovery is increased higher than control.
 28. The processof claim 21, wherein liquefying step (a) is performed at a temperaturein the range of 80° C.-90° C.
 29. The process of claim 21, whereinliquefying step (a) is performed at a pH in the range of 4-7.
 30. Theprocess of claim 21, wherein the alpha-amylase is a bacterialalpha-amylase.
 31. The process of claim 21, wherein the alpha-amylase isderived from Bacillus.
 32. The process of claim 21, whereinsaccharifying step (b) and fermenting step (c) are performedsimultaneously as a simultaneous saccharification and fermentation(SSF).
 33. The process of claim 32, wherein the SSF is performed at atemperature in the range of 25° C.-40° C.
 34. The process of claim 21,wherein the saccharification is performed at a pH in the range of 4-5.35. The process of claim 21, wherein the starch-containing material iscereal.
 36. The process of claim 21, wherein the starch-containingmaterial is selected from the group consisting of corn, wheat, barley,cassava, sorghum, rye, potato, beans, milo, peas, rice, sago, sweetpotatoes, tapioca, or any combination thereof.
 37. The process of claim21, wherein the starch-containing material comprises corn.
 38. Theprocess of claim 21, wherein the fermentation product is ethanol. 39.The process of claim 21, wherein the fermentation product is fuelethanol.
 40. The process of claim 21, wherein the fermenting organism isyeast.